PARG, a GTPase activating protein which interacts with PTPL1

ABSTRACT

The invention describes nucleic acids encoding the PARG protein, including fragments and biologically functional variants thereof. Also included are polypeptides and fragments thereof encoded by such nucleic acids, and antibodies relating thereto. Methods and products for using such nucleic acids and polypeptides also are provided.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/080,855,filed May 18, 1998, now issued as U.S. Pat. No. 6,083,721, which is acontinuation of application Ser. No. 08/805,583, filed Feb. 25, 1997,now abandoned.

FIELD OF THE INVENTION

This invention relates to nucleic acids and encoded polypeptides whichinteract with the PTPL1 phosphatase and which are GTPase activatingproteins. The invention also relates to agents which bind the nucleicacids or polypeptides. The invention further relates to methods of usingsuch nucleic acids and polypeptides in the treatment and/or diagnosis ofdisease.

BACKGROUND OF THE INVENTION

The Rho family of Ras-like GTPases, which includes Rho, Rac and Cdc42,control actin-based cytoskeletal rearrangements (reviewed in Hall, Annu.Rev. Cell Biol. 10:31-54, 1994; Zigmond, Curr. Opin. Cell Biol. 8:66-73,1996). Rho regulates receptor-mediated assembly of focal adhesions andstress fibers (Ridley and Hall, Cell 70:389-399, 1992), while Racregulates the formation of membrane ruffles (Ridley et al., Cell70:401-410, 1992) and Cdc42 controls the formation of filopodia (Nobesand Hall, Cell 81:53-62, 1995). Rho proteins have also been shown to beimportant in the regulation of cell proliferation (reviewed in Symons,Trends Biochem. Sci. 21:178-181, 1996). As members of the Rassuperfamily, Rho proteins function as molecular switches, having anactive, GTP-bound form, and an inactive, GDP-bound form. The active,GTP-bound form, is negatively regulated by GTPase activating proteins(GAPs) which enhance the intrinsic GTPase activity of Rho proteins. Anumber of GAPs that are active on proteins of the Rho family have beenidentified (reviewed in Lamarche and Hall, TIG 10:436-440, 1994). Theseinclude p50RhoGAP (Lancaster et al., J. Biol. Chem. 269:1137-1142,1994), Myr5 (Reinhard et al., EMBO J. 14:697-704, 1995), and p190(Settleman et al., Nature 359:153-154, 1992) which are also active onRac and Cdc42. Another GAP, p122-RhoGAP (Homma and Emori, EMBO J.14:286-291, 1995) appears to be specific for Rho.

Intracellular protein tyrosine phosphatases (PTPs) are a diverse groupof proteins involved in signal transduction (reviewed in Streuli, Curr.Opin. Cell Biol. 8:182-188, 1996). They contain a conserved PTP domainwhich specifically dephosphorylates tyrosine residues and, in addition,domains that regulate their subcellular localization and activity(reviewed in Mauro and Dixon, Trends Biochem. Sci. 19:151-155, 1994).For example, the SH2 domains of SHP-1 and SHP-2 enables these PTPs tolocalize to and interact with activated growth factor receptors (Mauroand Dixon, 1994). Correct localization of PTPs is of importance, sincethe PTP domains usually have broad substrate specificity.

PTPL1 (Saras et al., J. Biol. Chem. 269:24082-24089, 1994) also calledPTP-BAS (Maekawa et al., FEBS Lett. 337:200-206, 1994), hPTP1E (Banvilleet al., J. Biol. Chem. 269:22320-22327, 1994) and FAP-1 (Sato et al.,Science 268:411-415, 1995), is a 250 kDa protein expressed in manytissues and cell lines. PTPL1 is fully described in PCT publishedapplication WO95/06735. It contains an N-terminal leucine zipper motiffollowed by a domain with homology to the Band 4.1 superfamily. Band4.1-like domains are found in proteins involved in the linkage of actinfilaments to the plasma membrane (Arpin et al., Curr. Opin. Cell Biol.6:136-141, 1994). Five PDZ domains [PDZ is derived from PSD-95 (Cho etal., Neuron 9:929-942, 1992), Dlg-A (Woods and Bryant, Cell 66:451-464,1991) and ZO-1 (Itoh et al., J. Cell. Biol. 121:491-502, 1993), each ofwhich contains three such domains] are present between the Band 4.1-likedomain and the C-terminal PTP domain. These domain structures of about90 amino acid residues have also been called GLGF repeats or DHRs andare identified in a variety of proteins (Ponting and Phillips, TrendsBiochem. Sci. 20:102-103, 1995). A PDZ domain of PTPL1 has been shown tointeract with the C-terminal tail of the membrane receptor Fas (Sato etal., 1995) and PDZ domains of PSD-95 bind to the C-terminals of theNMDA-receptor and Shaker-type K⁺ channels (Kim et al., Nature 378:85-88,1995; Kornau et al., Science 269:1737-1740, 1995). The crystalstructures of two PDZ domains have recently been published (Doyle etal., Cell 85:1067-1076, 1996; Morais Cabral et al., Nature 382:649-652,1996).

There exists a need to influence the receptor-mediated intracellularsignal transduction pathways to treat disease. There also exists a needto identify the gene(s) responsible for increased or decreased signaltransduction and to provide a genetic therapy for treating diseasesresulting from aberrant signal transduction.

An object of the invention is to provide compounds that desirablyinfluence the signal transduction by the Rho family of Ras-like GTPases.

Another object of the invention is to provide therapeutics for treatingdiseases resulting from aberrant signal transduction by the Rho familyof Ras-like GTPases.

Still another object of the invention is to provide diagnostics andresearch tools relating to PARG, PTPL1 and the Rho family of Ras-likeGTPases. These and other objects will be described in greater detailbelow.

SUMMARY OF THE INVENTION

The invention provides isolated nucleic acid molecules, unique fragmentsof those molecules, expression vectors containing the foregoing, andhost cells transfected with those molecules. The invention also providesisolated polypeptides and agents which bind such polypeptides, includingantibodies. The foregoing can be used in the diagnosis or treatment ofconditions characterized by the expression of a PARG nucleic acid orpolypeptide. The invention also provides methods for identifyingpharmacological agents useful in the diagnosis or treatment of suchconditions. Here, we present the cDNA cloning of a PTPL1-associatedRhoGAP, PARG, a 150 kDa protein that contains a GAP domain that displaysstrong activity towards Rho. Furthermore, the C-terminal tail of PARGspecifically interacts with the fourth PDZ domain (PDZ4) of PTPL1.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The molecule hybridizes under stringent conditionsto a molecule consisting of the nucleic acid sequence of SEQ ID NO:1.The isolated nucleic acid molecule codes for a GTPase activatingpolypeptide. The invention further embraces nucleic acid molecules thatdiffer from the foregoing isolated nucleic acid molecules in codonsequence due to the degeneracy of the genetic code. The invention alsoembraces complements of the foregoing nucleic acids.

In preferred embodiments, the isolated nucleic acid molecule comprises amolecule consisting of the nucleic acid sequence of SEQ ID NO:1. Morepreferably, the isolated nucleic acid molecule comprises a moleculeconsisting of nucleotides 184-3966 of SEQ ID NO:1. Preferably theisolated nucleic acid comprises a molecule having a sequence whichencodes amino acids 666-853 of SEQ ID NO:2, amino acids 613-652 of SEQID NO:2, and/or amino acids 193-509 of SEQ ID NO:2.

According to another aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule comprises amolecule consisting of a unique fragment of nucleotides 184-3966 of SEQID NO:1 between 12 and 3781 nucleotides in length and complementsthereof, provided that the isolated nucleic acid molecule excludesmolecules consisting solely of nucleotide sequences selected from thegroup consisting of accession numbers T32345 (SEQ ID NO:3), Z28937 (SEQID NO:4), Z28520 (SEQ ID NO:5), AA431926 (SEQ ID NO:14), AA326126 (SEQID NO:15), AA342471 (SEQ ID NO:16), AA716829 (SEQ ID NO:17), L49573,Z43348 (SEQ ID NO:18), AA303722 (SEQ ID NO:19), T32495 (SEQ ID NO:20),AA330162 (SEQ ID NO:21), Z25350 (SEQ ID NO:22), AA794256 (SEQ ID NO:23),T32506 (SEQ ID NO:24), T32263 (SEQ ID NO:25), F06673 (SEQ ID NO:26),AA462548 (SEQ ID NO:27), X85558 (SEQ ID NO:28), R14952 (SEQ ID NO:29),AA870705 (SEQ ID NO:30), AA120493 (SEQ ID NO:3 1), AA415591 (SEQ IDNO:32), AA1 31400 (SEQ ID NO:33), C76597 (SEQ ID NO:34), C76601 (SEQ IDNO:35), AA870475 (SEQ ID NO:36), AA234871 (SEQ ID NO:37), C77518 (SEQ IDNO:38), and AA672012 (SEQ ID NO:39). In one embodiment, the isolatednucleic acid molecule consists of between 12 and 32 contiguousnucleotides of SEQ ID NO:1, or complements of such, nucleic acidmolecules. In preferred embodiments, the unique fragment is at least 14,15, 16, 17, 18, 20 or 22 contiguous nucleotides of the nucleic acidsequence of SEQ ID NO:1, or complements thereof.

According to another aspect of the invention, an isolated nucleic acidmolecule which encodes a PDZ domain binding site is provided, comprisinga sequence selected from the group consisting of SEQ ID NO:6, SEQ IDNO:8 and SEQ ID NO:10, or nucleic acid molecules that differ from thenucleic acid molecules of the group consisting of SEQ ID NO:6, SEQ IDNO:8 and SEQ ID NO:10 in codon sequence due to the degeneracy of thegenetic code. Preferably the isolated nucleic acid consists of amolecule having a sequence selected from the group consisting of SEQ IDNO:6, SEQ ID NO:8 and SEQ ID NO:10.

According to another aspect of the invention, the invention involvesexpression vectors, and host cells transformed or transfected with suchexpression vectors, comprising the nucleic acid molecules describedabove.

According to another aspect of the invention, an isolated polypeptide isprovided. The isolated polypeptide is encoded by the isolated nucleicacid molecule, and the polypeptide has GTPase activating activity. Inpreferred embodiments, the isolated polypeptide comprises a polypeptidehaving the sequence of amino acids 658-898 of SEQ ID NO:2.

According to a further aspect of the invention, an isolated polypeptideis provided. The isolated polypeptide comprises a polypeptide encoded bya nucleic acid which hybridizes under stringent conditions tonucleotides 2020-2139 of SEQ ID NO:1. In preferred embodiments, theisolated polypeptide comprises a polypeptide having the sequence ofamino acids 613-652 of SEQ ID NO:2 is provided. The isolated polypeptidehas a Cys-rich domain.

According to another aspect of the invention, an isolated polypeptide isprovided. The isolated polypeptide comprises a polypeptide encoded by anucleic acid which hybridizes under stringent conditions to nucleotides760-1710 of SEQ ID NO:1. In preferred embodiments, the isolatedpolypeptide comprises a polypeptide having the sequence of amino acid193-509 of SEQ ID NO:2 is provided. The isolated polypeptide is a ZPHdomain polypeptide.

In other embodiments, the isolated polypeptide consists of a fragment orvariant of the foregoing which retains the activity of the foregoing.

According to still another aspect of the invention, an isolatedpolypeptide is provided. The isolated polypeptide is encoded by anucleic acid molecule having a sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10. The isolatedpolypeptide comprises a polypeptide selected from the group consistingof a polypeptide having the sequence of SEQ ID NO:7, a polypeptidehaving the sequence of SEQ ID NO:9, and a polypeptide having thesequence of SEQ ID NO:11.

According to another aspect of the invention, there are providedisolated polypeptides which selectively bind a PARG protein or fragmentthereof. The isolated polypeptide in certain embodiments binds to apolypeptide comprising the sequence of amino acids 658-898 of SEQ IDNO:2, amino acids 613-652 of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11 or amino acids 193-509 of SEQ ID NO:2. The isolated polypeptidepreferably binds to a polypeptide consisting essentially of the sequenceof amino acids 658-898 of SEQ ID NO:2, amino acids 613-652 of SEQ IDNO:2, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or amino acids 193-509 ofSEQ ID NO:2. In preferred embodiments, isolated binding-polypeptidesinclude antibodies and fragments of antibodies (e.g., Fab, F(ab)₂, Fdand antibody fragments which include a CDR3 region which bindsselectively to the PARG polypeptides of the invention).

The invention provides in another aspect an isolated complex ofpolypeptides. The isolated complex includes a PTPL1 polypeptide, such apolypeptide including the amino acid sequence of SEQ ID NO:12 bound to apolypeptide as claimed in claim 1. The isolated complex has both PTPL1phosphatase activity and PARG GAP activity. Preferably the isolatedcomplex consists essentially of the polypeptide of SEQ ID NO:12 and thepolypeptide of SEQ ID NO:2.

According to still another aspect of the invention, methods for reducingRho family GTPase signal transduction in a mammalian cell are provided.The methods involve administering to a mammalian cell an amount of aninhibitor of Rho family GTPase activity effective to reduce Rho familyGTPase signal transduction in the mammalian cell. In certainembodiments, the inhibitor is an isolated PARG polypeptide, having RhoGAP activity, encoded by SEQ ID NO:1. In other embodiments, theinhibitor is an isolated complex of polypeptides comprising apolypeptide comprising the amino acid sequence of SEQ ID NO:12 and apolypeptide comprising the amino acid sequence of SEQ ID NO:2.

According to still another aspect of the invention, methods for reducingproliferation of a cancer cell are provided. The methods involveadministering to a cancer cell an amount of a PARG polypeptide,comprising a polypeptide encoded by the nucleic acid of claim 1,effective to reduce proliferation of the cancer cell.

The invention in a further aspect provides methods for increasing Rhofamily GTPase signal transduction in a mammalian cell. A dominantnegative variant of the polypeptide of SEQ ID NO:2 is administered tothe mammalian cell in an amount effective to increase Rho family GTPasesignal transduction. Preferably the dominant negative polypeptideincludes an inactivated GTPase activating domain which contains adeletion or at least one inactivating point mutation.

According to a further aspect of the invention, methods for reducingbinding of a protein which includes a PDZ4 domain to a protein whichincludes a PDZ4 domain binding site are provided. The methods involvecontacting the protein which includes PDZ4 domain with an agent whichbinds to the PDZ4 domain for a time effective to reduce the binding ofthe protein which includes PDZ4 domain to the protein which includesPDZ4 domain binding site. In certain embodiments the agent is anisolated peptide and includes at its carboxyl terminus the amino acidsequence of SEQ ID NO:7. The isolated peptide can include conservativesubstitutions of the amino acid sequence of SEQ ID NO:7, excepting theterminal valine. In preferred embodiments the amino acid sequence of thepeptide is selected from the group consisting of SEQ ID NO:7, SEQ IDNO:9 and SEQ ID NO:11. In other embodiments the agent is an antibodywhich binds to the PDZ4 domain, preferably a monoclonal antibody. Insome embodiments, methods provide inhibiting binding of a protein whichincludes a PDZ4domain and a protein which includes a PDZ4domain bindingsite in a mammalian cell. Such methods involve contacting the mammaliancell with an agent which binds to the PDZ4 domain for a time effectiveto reduce the binding of the protein which includes PDZ4 domain to theprotein which includes PDZ4 domain binding site.

The invention in another aspect provides methods of modulating mast cellsecretion in a subject. The methods include administering to the subjectin need of such treatment an amount of a modulator of PARG GTPaseactivating activity effective to modulate mast cell secretion in thesubject.

The invention in still another aspect provides compositions comprising aPARG polypeptide which has GTPase activating activity, a complex of sucha PARG polypeptide and PTPL1 phosphatase, or a peptide agent which bindsto a PDZ4 domain and which includes the sequence of SEQ ID NO:7, and apharmaceutically acceptable carrier.

The invention in a further aspect involves a method for decreasing PARGGTPase activating activity in a subject. An agent that selectively bindsto an isolated nucleic acid molecule of the invention or an expressionproduct thereof is administered to a subject in need of such treatment,in an amount effective to decrease PARG GTPase activating activity inthe subject. Preferred agents are antisense nucleic acids, includingmodified nucleic acids, and polypeptides.

According to another aspect of the invention, methods are provided foridentifying lead compounds for a pharmacological agent useful in thediagnosis or treatment of disease associated with PARG GTPase activatingactivity or with PARG binding to a protein containing a PDZ4 domain. Themethods involve forming a mixture of a PARG polypeptide or fragmentthereof containing a GTPase activating domain or a PDZ4 domain bindingsite, a protein which interacts with the foregoing GTPase activatingdomain or PDZ4 domain binding site, and a candidate pharmacologicalagent. The mixture is incubated under conditions which, in the absenceof the candidate pharmacological agent, permit a first amount ofspecific activation of the GTPase by the PARG GTPase activating domainor permit a first amount of selective binding of the protein containinga PDZ4 domain by the PDZ4 domain binding site. A test amount of thespecific activation of the GTPase by the PARG GTPase activating domainor the selective binding of the protein containing a PDZ4 domain by thePDZ4 domain binding site then is detected. Detection of an increase inthe foregoing activities in the presence of the candidatepharmacological agent indicates that the candidate pharmacological agentis a lead compound, for a pharmacological agent which increases specificactivation of the GTPase by the PARG GTPase activating domain orselective binding of the protein containing a PDZ4 domain by the PDZ4domain binding site. Detection of a decrease in the foregoing activitiesin the presence of the candidate pharmacological agent indicates thatthe candidate pharmacological agent is a lead compound for apharmacological agent which decreases specific activation of the GTPaseby the PARG GTPase activating domain or selective binding of the proteincontaining a PDZ4 domain by the PDZ4 domain binding site. Where theactivity tested is specific activation of the GTPase, the protein whichinteracts with the GTPase activating domain preferably is Rho. Where theactivity tested is selective binding of a PDZ4 domain, the protein whichinteracts with the PDZ4 domain binding site preferably is PTPL1.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are a representation of the production GST-PDZ fusionproteins. (A) Schematic illustration of the GST-PDZ fusion proteinsshowing the domain structure of PTPL1 and the design of PTPL1-derivedGST-PDZ fusion proteins (B) Expression of GST-PDZ fusion proteins.

FIG. 2 shows the interaction of GST-PDZ fusion proteins with componentsin cell lysate.

FIGS. 3A-3C depict the structure of PARG protein. (A) Deduced amino acidsequence of PARG (SEQ ID NO:2). (B) Comparison of amino acid sequencesof ZPH regions found in PARG (SEQ ID NO:2) and in the gene product ofthe C. elegans gene ZK669.1a (SEQ ID NO:13). (C) Schematic diagramillustrating the domain structure of PARG and ZK669.1a.

FIG. 4 shows Northern blot analysis of expression of PARG mRNA indifferent human tissues.

FIGS. 5A-5D show an analysis of the GAP activity of PARG. (A) Expressionof the GAP domain of PARG as a GST fusion protein. Rho (B), Rac (C), andCdc42 (D) loaded with γ-³²P-GTP were incubated with 1 nM (open circles),20 nM (filled circles) of the GAP domain of PARG expressed as a GSTfusion protein, or 100 nM GST (squares) as a control, for different timeperiods at 30° C.

FIG. 6 shows binding of GST-PDZ fusion proteins to a C-terminal PARGpeptide.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of the PARG cDNA.

SEQ ID NO:2 is the amino acid sequence of the translation product of thePARG cDNA, including a RhoGAP domain at amino acids 666-853, acysteine-rich domain at amino acids 613-652, a ZPH domain at amino acids193-509 of SEQ ID NO:2, and a carboxyl-terminal PDZ domain binding site.

SEQ ID NO:3 is the nucleotide sequence of the expressed sequence tagidentified by GenBank accession number T32345.

SEQ ID NO:4 is the nucleotide sequence of the expressed sequence tagidentified by GenBank accession number Z28937.

SEQ ID NO:5 is the nucleotide sequence of the expressed sequence tagidentified by GenBank accession number Z28520.

SEQ ID NO:6 is the nucleotide sequence encoding the PARG PDZ domainbinding site. which consists of 4 amino acids.

SEQ ID NO:7 is the amino acid sequence of the PARG PDZ domain bindingsite which consists of 4 amino acids.

SEQ ID NO:8 is the nucleotide sequence encoding the PARG PDZ domainbinding site which consists of 5 amino acids.

SEQ ID NO:9 is the amino acid sequence of the PARG PDZ domain bindingsite which consists of 5 amino acids.

SEQ ID NO:10 is the nucleotide sequence encoding the PARG PDZ domainbinding site which consists of 6 amino acids.

SEQ ID NO:11 is the amino acid sequence of the PARG PDZ domain bindingsite which consists of 6 amino acids.

SEQ ID NO:12 is the amino acid sequence of the PTPL1 phosphatase.

SEQ ID NO:13 is a portion of the amino acid sequence of the ZK669.1aprotein (GenBank accession number Z37093).

BRIEF DESCRIPTION OF THE INVENTION

The present invention in one aspect involves the cloning of a cDNAencoding a PARG GTPase activating protein. The sequence of the humangene is presented as SEQ ID NO:1, and the predicted amino acid sequenceof this gene's protein product is presented as SEQ ID NO:2. Analysis ofthe sequence by comparison to nucleic acid and protein databasesdetermined that PARG has several domains in addition to the GAP domain.These include a cysteine-rich domain located directly N-terminal of theGAP domain, a ZPH domain similar to the ZK669.1 gene product of C.elegans (Wilson et al., Nature 368:32-38, 1994), and a PDZ domainbinding site.

The GAP activity of PARG was determined as reported in Example 7 below.The GAP activity of this protein is strongest on Rho GTPase in vitro.GAP activities were also detected on Rac and Cdc42 in vitro. Becausethese activities on Rac and Cdc42 were observed at higher PARGconcentrations than needed for Rho GAP activity, it is likely that Rhois the preferred in vivo target of PARG.

A cysteine-rich domain is located directly N-terminal of the GAP domainof PARG. This domain has been identified in various proteins includingmost PKC isoforms (which have two copies each of the domain), theprotooncogene products Vav and Raf, diacylglycerol kinase and chimaerins(reviewed by Newton, Curr. Biol. 5: 973-976, 1995). The cysteine-richdomain has been shown to bind Zn²⁺ (Ahmed et al., Biochem J. 280:233-241, 1991), and the domains found in PKCs and in chimaerins alsobind phorbol esters and diacylglycerol (Ahmed et al., 1991; Ono et al.,Proc. Natl. Acad. Sci. USA 86: 4868-4871, 1989). Generation ofdiacylglycerol or addition of phorbol ester increase the affinity of PKCmolecules for membranes, and the resulting translocation of PKC from thecytosol to the plasma membrane is likely to involve interactions betweenthe cysteine-rich domains and membrane phospholipids (Newton, 1995;Zhang et al., Cell 81: 917-924, 1995). The cysteine-rich domain of PARGmay mediate regulatable binding to the membrane and could possibly alsobe involved in regulation of the GAP activity. Thus, a function of thecysteine-rich domain of PARG may be analogous to a function ofn(α1)-chimaerin, a Rac-specific GAP, which contains a copy of ahomologous cysteine-rich domain; it has been shown that phospholipidsand phorbol esters regulate the GAP activity of n(α1)-chimaerin (Ahmedet al., J. Biol. Chem. 268: 10709-10712, 1993).

In the N-terminal part of PARG, a region of about 300 amino acidresidues with similarity (27% identity) to the gene product of the C.elegans gene ZK669.1a was identified, and denoted ZPH region. Theoverall domain structure of the ZK669.1 a gene product is similar toPARG and it is possible that PARG is the human homolog of the C. elegansZK669.1 a gene product. However, the RhoGAP domain and the cysteine-richdomain of the ZK669.1 a gene product is not significantly more similarto PARG (29% identity within the RhoGAP domains, 24% idenity within thecysteine-rich domains) compared to other human proteins containing thesedomains (24-31% identity within the RhoGAP domains and 16-27% identitywithin the cysteine-rich domains).

PDZ domains have been identified in a diverse set of proteins (Pontingand Phillips, Trends Biochem. Sci. 20: 102-103, 1995). These proteinsseem to be involved in signal transduction, and many of them, if notall, are found in structures at the plasma membrane. The size of the PDZdomain of about 90 amino acid residues, and its appearance in signaltransduction proteins suggested that it, like SH2 and SH3 domains, canmediate direct interactions with other molecules. We have shown thatPARG binds specificially to PDZ4 of PTPL1 and that the binding-site forbinding to PDZ 4 resides in the four most C-terminal amino acid residuesof PARG. PDZ domains can bind strongly to a short peptide of only fouramino acid residues, and the carboxy-group and the side chain of theC-terminal valine residue is important for binding. The crystalstructure of the third PDZ domain of PSD-95 binding to a peptide (Doyleet al., 1996; Morais Cabral et al., 1996) confirms these results andshows that the last four C-terminal amino acid residues of the peptidebind in a cleft of the domain with the C-terminal valine buried in ashallow pocket. Thus, the PDZ domain functions as a C-terminal peptidebinding module. Because PDZ 4 binds to PARG, a complex between PTPL1,PARG, and Rho can be formed. Protein tyrosine kinases have beenimplicated to act upstream and downstream of Rho (Nobes and Hall, J.Cell Sci. 108:225-233, 1995; Ridley, BioEssays 16:321-327, 1994). Thus,PTPL1 can function as a negative regulator of kinases in the Rho signalpathway, and in complex with PARG, which inactivates Rho itself, it canbe a powerful inhibitor of Rho signals.

The invention thus involves in one aspect PARG polypeptides, genesencoding those polypeptides, functional modifications and variants ofthe foregoing, useful fragments of the foregoing, as well astherapeutics relating thereto.

Homologs and alleles of the PARG nucleic acids of the invention can beidentified by conventional techniques. Thus, an aspect of the inventionis those nucleic acid sequences which code for PARG polypeptides andwhich hybridize to a nucleic acid molecule consisting of the codingregion of SEQ ID NO:1, under stringent conditions. The term “stringentconditions” as used herein refers to parameters with which the art isfamiliar. Nucleic acid hybridization parameters may be found inreferences which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, stringentconditions, as used herein, refers, for example, to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄(pH7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS issodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.After hybridization, the membrane upon which the DNA is transferred iswashed at 2×SSC at room temperature and then at 0.1×SSC/0.1×SDS attemperatures up to 65° C.

There are other conditions, reagents, and so forth which can used, whichresult in a similar degree of stringency. The skilled artisan will befamiliar with such conditions, and thus they are not given here. It willbe understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of PARG nucleic acids of the invention. Theskilled artisan also is familiar with the methodology for screeningcells and libraries for expression of such molecules which then areroutinely isolated, followed by isolation of the pertinent nucleic acidmolecule and sequencing.

In general homologs and alleles typically will share at least 40%nucleotide identity and/or at least 50% amino acid identity to SEQ IDNO:1 and SEQ ID NO:2, respectively, in some instances will share atleast 50% nucleotide identity and/or at least 65% amino acid identityand in still other instances will share at least 60% nucleotide identityand/or at least 75% amino acid identity. Watson-Crick complements of theforegoing nucleic acids also are embraced by the invention.

In screening for PARG proteins, a Southern blot may be performed usingthe foregoing conditions, together with a radioactive probe. Afterwashing the membrane to which the DNA is finally transferred, themembrane can be placed against X-ray film to detect the radioactivesignal.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating PARGpolypeptide. Similarly, nucleotide sequence triplets which encode otheramino acid residues include, but are not limited to,: CCA, CCC, CCG andCCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons);ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparaginecodons); and ATA, ATC and ATT (isoleucine codons). Other amino acidresidues may be encoded similarly by multiple nucleotide sequences.Thus, the invention embraces degenerate nucleic acids that differ fromthe biologically isolated nucleic acids in codon sequence due to thedegeneracy of the genetic code.

The invention also provides isolated unique fragments of SEQ ID NO:1 orcomplements of SEQ ID NO:1. A unique fragment is one that is a‘signature’ for the larger nucleic acid. It, for example, is long enoughto assure that its precise sequence is not found in molecules outside ofthe PARG nucleic acids defined above. Unique fragments can be used asprobes in Southern blot assays to identify such nucleic acids, or can beused in amplification assays such as those employing PCR. As known tothose skilled in the art, large probes such as 200, 250, 300, 400, 500nucleotides or more are preferred for certain uses such as Southernblots, while smaller fragments will be preferred for uses such as PCR.Unique fragments also can be used to produce fusion proteins forgenerating antibodies or determining binding of the polypeptidefragments, as demonstrated in the Examples, or for generatingimmunoassay components. Likewise, unique fragments can be employed toproduce nonfused fragments of the PARG polypeptides, useful, forexample, in the preparation of antibodies, in immunoassays, and as acompetitive binding partner of the PTPL1 phosphatase and/or otherpolypeptides which bind to the PARG polypeptides, for example, intherapeutic applications. Unique fragments further can be used asantisense molecules to inhibit the expression of PARG nucleic acids andpolypeptides, particularly for therapeutic purposes as described ingreater detail below.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NO:1 and its complement will require longersegments to be unique while others will require only short segments,typically between 12 and 32 nucleotides (e.g. 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 baseslong). Virtually any segment of the region of SEQ ID NO:1 beginning atnucleotide 184 and ending at nucleotide 3966, or its complement, that is18 or more nucleotides in length will be unique. Those skilled in theart are well versed in methods for selecting such sequences, typicallyon the basis of the ability of the unique fragment to selectivelydistinguish the sequence of interest from non-PARG nucleic acids. Acomparison of the sequence of the fragment to those on known data basestypically is all that is necessary, although in vitro confirmatoryhybridization and sequencing analysis may be performed. Thus, forexample, an examination of the nucleotide sequence databases indicatesthat at least a portion of the following sequences are identical to thePARG sequence, and thus nucleic acid molecules consisting solely of thefollowing nucleotide sequences are not unique fragments of PARG:AA431926, AA326126, AA342471, AA716829, L49573, Z43348, AA303722,Z28520, T32495, AA330162, Z25350, AA794256, T32506, T32263, F06673,T32345, Z28937, AA462548, X85558, R14952, AA870705AA120493, AA415591,AA131400, C76597, C76601, AA870475, AA234871, C77518, and AA672012.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding a PARGpolypeptide, to decrease GTPase activation by PARG or phosphatasebinding by PARG. This is desirable in virtually any medical conditionwherein a reduction in GTPase activating activity of PARG is desirable,including to reduce Rho family protein signal transduction, or wherein areduction in phosphatase binding by PARG is desirable. Antisensemolecules, in this manner, can be used to slow down or arrest theproliferation of cancer cells in vivo.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon SEQ ID NO:1, or upon allelic or homologousgenomic and/or cDNA sequences, one of skill in the art can easily chooseand synthesize any of a number of appropriate antisense molecules foruse in accordance with the present invention. In order to besufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 10 and, more preferably, atleast 15 consecutive bases which are complementary to the target,although in certain cases modified oligonucleotides as short as 7 basesin length have been used successfully as antisense oligonucleotides(Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably,the antisense oligonucleotides comprise a complementary sequence of20-30 bases. Although oligonucleotides may be chosen which are antisenseto any region of the gene or mRNA transcripts, in preferred embodimentsthe antisense oligonucleotides correspond to N-terminal or 5′ upstreamsites such as translation initiation, transcription initiation orpromoter sites. In addition, 3′-untranslated regions may be targeted.Targeting to mRNA splicing sites has also been used in the art but maybe less preferred if alternative mRNA splicing occurs. In addition, theantisense is targeted, preferably, to sites in which mRNA secondarystructure is not expected (see, e.g., Sainio et al., Cell Mol.Neurobiol. 14(5):439-457, 1994) and at which proteins are not expectedto bind. Finally, although, SEQ ID NO:1 discloses a cDNA sequence, oneof ordinary skill in the art may easily derive the genomic DNAcorresponding to the cDNA of SEQ ID NO:1. Thus, the present inventionalso provides for antisense oligonucleotides which are complementary tothe genomic DNA corresponding to SEQ ID NO:1. Similarly, antisense toallelic or homologous cDNAs and genomic DNAs are enabled without undueexperimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisense molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acids encoding PARGpolypeptides, together with pharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate in a host cell, and which is further characterized by one ormore endonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques (e.g., green fluorescent protein). Preferredvectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which theyare operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding PARG polypeptide or fragment or variantthereof. That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen), which contains an Epstein Barr virus (EBV) originof replication, facilitating the maintenance of plasmid as a multicopyextrachromosomal element. Another expression vector is the pEF-BOSplasmid containing the promoter of polypeptide Elongation Factor 1α,which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdisclosed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J. Cancer, 67:303-310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

The invention also permits the construction of PARG gene “knock-outs” incells and in animals, providing materials for studying certain aspectsof GTPase activating activity and signal transduction.

The invention also provides isolated polypeptides, which include thepolypeptide of SEQ ID NO:2 and unique fragments of SEQ ID NO:2,particularly amino acids 193-509, 613-652 and 658-898 of SEQ ID NO:2, aswell as the carboxyl terminal 4, 5 or 6 amino acids of SEQ ID NO:2. Suchpolypeptides are useful, for example, alone or as fusion proteins togenerate antibodies, as a components of an immunoassay.

A unique fragment of an PARG polypeptide, in general, has the featuresand characteristics of unique fragments as discussed above in connectionwith nucleic acids. As will be recognized by those skilled in the art,the size of the unique fragment will depend upon factors such as whetherthe fragment constitutes a portion of a conserved protein domain. Thus,some regions of amino acids 658-898 of SEQ ID NO:2, amino acid residues613-652 of SEQ ID NO:2 and amino acid residues of 193-509 SEQ ID NO:2,will require longer segments to be unique while others will require onlyshort segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8,9, 10, 11 and 12 amino acids long). Virtually any segment of amino acids658-898 of SEQ ID NO:2, amino acid residues 613-652 of SEQ ID NO:2 andamino acid residues of 193-509 SEQ ID NO:2, that is 10 or more aminoacids in length will be unique.

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptides(such as Rho) or fragments thereof, selective binding of nucleic acidsor proteins (such as PTPL1), and enzymatic activity. Those skilled inthe art are well versed in methods for selecting unique amino acidsequences, typically on the basis of the ability of the unique fragmentto selectively distinguish the sequence of interest from non-familymembers. A comparison of the sequence of the fragment to those on knowndata bases typically is all that is necessary.

The invention embraces variants of the PARG polypeptides describedabove. As used herein, a “variant” of a PARG polypeptide is apolypeptide which contains one or more modifications to the primaryamino acid sequence of a PARG polypeptide. Modifications which create aPARG variant can be made to a PARG polypeptide 1) to reduce or eliminatean activity of a PARG polypeptide, such as PTPL1 binding or GAP activityfor Rho GTPase; 2) to enhance a property of a PARG polypeptide, such asprotein stability in an expression system or the stability ofprotein-protein binding; or 3) to provide a novel activity or propertyto a PARG polypeptide, such as addition of an antigenic epitope oraddition of a detectable moiety. Modifications to a PARG polypeptide aretypically made to the nucleic acid which encodes the PARG polypeptide,and can include deletions, point mutations, truncations, amino acidsubstitutions and additions of amino acids or non-amino acid moieties.Alternatively, modifications can be made directly to the polypeptide,such as by cleavage, addition of a linker molecule, addition of adetectable moiety, such as biotin, addition of a fatty acid, and thelike. Modifications also embrace fusion proteins comprising all or partof the PARG amino acid sequence.

In general, variants include PARG polypeptides which are modifiedspecifically to alter a feature of the polypeptide unrelated to itsphysiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of aPARG polypeptide by eliminating proteolysis by proteases in anexpression system (e.g., dibasic amino acid residues in yeast expressionsystems in which KEX2 protease activity is present).

Mutations of a nucleic acid which encode a PARG polypeptide preferablypreserve the amino acid reading frame of the coding sequence, andpreferably do not create regions in the nucleic acid which are likely tohybridize to form secondary structures, such a hairpins or loops, whichcan be deleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant PARG polypeptides) which are silent as tothe amino acid sequence of the polypeptide, but which provide preferredcodons for translation in a particular host. The preferred codons fortranslation of a nucleic acid in, e.g., E. Coli, are well known to thoseof ordinary skill in the art. Still other mutations can be made to thenoncoding sequences of a PARG gene or cDNA clone to enhance expressionof the polypeptide. The activity of variants of PARG polypeptides can betested by cloning the gene encoding the variant PARG polypeptide into abacterial or mammalian expression vector, introducing the vector into anappropriate host cell, expressing the variant PARG polypeptide, andtesting for a functional capability of the PARG polypeptides asdisclosed herein. For example, the variant PARG polypeptide can betested for Rho GAP activity as disclosed in Example 7, or for PDZbinding as disclosed in other Examples herein. Preparation of othervariant polypeptides may favor testing of other activities, as will beknown to one of ordinary skill in the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in PARG polypeptides to provide functionallyequivalent variants of the foregoing polypeptides, i.e, the variantsretain the functional capabilities of the PARG polypeptides. As usedherein, a “conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Exemplary functionally equivalentvariants of the PARG polypeptides include conservative amino acidsubstitutions of SEQ ID NO:2, particularly conservative substitutions ofamino acids other than 193-509, 613-652 or 658-898 of SEQ ID NO:2.However, conservative substitutions of amino acids 193-509, 613-652 or658-898 of SEQ ID NO:2 can be made as well. Conservative substitutionsof amino acids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D. Changes to the carboxyl terminalvaline of the PARG PDZ domain binding site are not preferred forretention of maximal binding activity.

Conservative amino-acid substitutions in the amino acid sequence ofPARG-polypeptides to produce functionally equivalent variants of PARGpolypeptides typically are made by alteration of the nucleic acidencoding PARG polypeptides (SEQ ID NO:1). Such substitutions can be madeby a variety of methods known to one of ordinary skill in the art. Forexample, amino acid substitutions may be made by PCR-directed mutation,site-directed mutagenesis according to the method of Kunkel (Kunkel,Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or by chemicalsynthesis of a gene encoding a PARG polypeptide. Where amino acidsubstitutions are made to a small unique fragment of a PARG polypeptide,such as a PDZ-domain binding site peptide, the substitutions can be madeby directly synthesizing the peptide. The activity of functionallyequivalent fragments of PARG polypeptides can be tested by cloning thegene encoding the altered PARG polypeptide into a bacterial or mammalianexpression vector, introducing the vector into an appropriate host cell,expressing the altered PARG polypeptide, and testing for a functionalcapability of the PARG polypeptides as disclosed herein. Peptides whichare chemically synthesized can be tested directly for function, e.g.,for binding to a PDZ 4 domain of PTPL1.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof the PARG protein molecule (SEQ ID NO:2). A variety of methodologieswell-known to the skilled practitioner can be utilized to obtainisolated PARG molecules. The polypeptide may be purified from cellswhich naturally produce the polypeptide by chromatographic means orimmunological recognition. Alternatively, an expression vector may beintroduced into cells to cause production of the polypeptide. In anothermethod, mRNA transcripts may be microinjected or otherwise introducedinto cells to cause production of the encoded polypeptide. Translationof mRNA in cell-free extracts such as the reticulocyte lysate systemalso may be used to produce polypeptide. Those skilled in the art alsocan readily follow known methods for isolating PARG polypeptides. Theseinclude, but are not limited to, immunochromotography, HPLC,size-exclusion chromatography, ion-exchange chromatography andimmune-affinity chromatography.

The isolation of the PARG gene also makes it possible for the artisan todiagnose a disorder characterized by expression of PARG. These methodsinvolve determining expression of the PARG gene, and/or PARGpolypeptides derived therefrom. In the former situation, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction asexemplified in the examples below, or assaying with labeledhybridization probes.

The invention also makes it possible isolate proteins having a PDZ4domain by the binding of such proteins to the PDZ domain binding sitedisclosed herein. The identification of the PDZ domain binding site alsopermits one of skill in the art to block the binding of a protein havinga PDZ4 domain, such as PTPL1, with a binding partner having a PDZ4domain binding site, such as PARG. Binding of the proteins can beeffected by introducing into a biological system in which the proteinsbind (e.g., a cell) a polypeptide including a PDZ domain binding site inan amount sufficient to block the binding. The identification of thePDZ4 domain binding site in PARG also enables one of skill in the art toprepare modified proteins, using standard recombinant DNA techniques,which can bind to proteins containing a PDZ4 domain. For example, whenone desires to target a certain protein to the inner membrane surfacewhere proteins containing a PDZ domain, such as PTPL1, are localized,one can prepare a fusion polypeptide of the protein and the PDZ4 domainbinding site. Preferably, the PDZ domain binding site is fused to thecarboxy terminus of the protein. Additional uses are described furtherherein.

The invention further provides methods for reducing or increasing Rhofamily signal transduction in a cell. Such methods are useful in vitrofor altering the Rho signal transduction, for example, in testingcompounds for potential to block aberrant Rho signal transduction. Invivo, such methods are useful for modulating actin polymerization, cellproliferation and release of secretory granules from mast cells (see,e.g., Price et al., Curr. Biol. 5:68-73, 1995), e.g., to treat allergy.Increasing Rho signal transduction in a cell by, e.g., introducing adominant negative PARG polypeptide in the cell, can be used to provide amodel system for testing the effects of putative inhibitors of Rhosignal transduction. Such methods also are useful in the treatment ofconditions which result from excessive or deficient Rho signaltransduction. Rho signal transduction can be measured by studying actinreorganization or by measuring the ratio of Rho-bound GTP/GDP. Variousmodulators of PARG GTPase activating activity can be screened foreffects on Rho signal transduction using the methods disclosed herein.The skilled artisan can first determine the modulation of a PARGactivity, such as GTPase activating activity, and then apply such amodulator to a target cell or subject and assess the effect on thetarget cell or subject. For example, in screeing for modulators of PARGuseful in the treatment of mast cell secretion, mast cells in culturecan be contacted with PARG modulators and the increase or decrease ofsecretory granule release by the mast cells can be determined accordingto standard procedures. PARG activity modulators can be assessed fortheir effects on other Rho signal transduction downstream effects bysimilar methods in other cell types.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from SEQ ID NO:2. A dominant negative polypeptideis an inactive variant of a protein, which, by interacting with thecellular machinery, displaces an active protein from its interactionwith the cellular machinery or competes with the active protein, therebyreducing the effect of the active protein. For example, a dominantnegative receptor which binds a ligand but does not transmit a signal inresponse to binding of the ligand can reduce the biological effect ofexpression of the ligand. Likewise, a dominant negativecatalytically-inactive kinase which interacts normally with targetproteins but does not phosphorylate the target proteins can reducephosphorylation of the target proteins in response to a cellular signal.Similarly, a dominant negative transcription factor which binds to apromoter site in the control region of a gene but does not increase genetranscription can reduce the effect of a normal transcription factor byoccupying promoter binding sites without increasing transcription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and using standard mutagenesis techniques tocreate one or more dominant negative variant polypeptides. For example,given the teachings contained herein of a PARG polypeptide, one ofordinary skill in the art can modify the sequence of the PARGpolypeptide by site-specific mutagenesis, scanning mutagenesis, partialgene deletion or truncation, and the like. See, e.g., U.S. Pat. No.5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilledartisan then can test the population of mutagenized polypeptides fordiminution in a selected activity (e.g., PARG GAP activity) and forretention of a desired activity (e.g., PARG binding to PTPL1). Othersimilar methods for creating and testing dominant negative variants of aprotein will be apparent to one of ordinary skill in the art.

Dominant negative PARG proteins include variants in which a portion ofthe PDZ4 domain binding site has been mutated or deleted to reduce oreliminate PARG interaction with PTPL1. Other examples include partialdeletion PARG variants which have the GAP domain deleted. Such variantsretain the capability to bind PTPL1 but cannot enhance GTPase activityin Rho. A GAP-negative PARG variant does not, therefore, stimulatedownstream signal transduction pathways such as the Rho pathway.

The invention also involves agents such as polypeptides which bind toPARG polypeptides and to complexes of PARG polypeptides and theirphosphatase binding partners. Such binding agents can be used, forexample, in screening assays to detect the presence or absence of PARGpolypeptides and complexes of PARG polypeptides and their phosphatasebinding partners and in purification protocols to isolate PARGpolypeptides and complexes of PARG polypeptides and their phosphatasebinding partners. Such agents also can be used to inhibit the nativeactivity of the PARG polypeptides or their phosphatase binding partners,for example, by binding to such polypeptides, or their binding partnersor both.

The invention, therefore, embraces peptide binding agents which, forexample, can be antibodies or fragments of antibodies having the abilityto selectively bind to PARG polypeptides. Antibodies include polyclonaland monoclonal antibodies, prepared according to conventionalmethodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to PARG polypeptides, and complexes of both PARGpolypeptides and their phosphatase binding partners. These polypeptidesmay be derived also from sources other than antibody technology. Forexample, such polypeptide binding agents can be provided by degeneratepeptide libraries which can be readily prepared in solution, inimmobilized form or as phage display libraries. Combinatorial librariesalso can be synthesized of peptides containing one or more amino acids.Libraries further can be synthesized of peptoids and non-peptidesynthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe PARG polypeptide. This process can be repeated through severalcycles of reselection of phage that bind to the PARG polypeptide.Repeated rounds lead to enrichment of phage bearing particularsequences. DNA sequence analysis can be conducted to identify thesequences of the expressed polypeptides. The minimal linear portion ofthe sequence that binds to the PARG polypeptide can be determined. Onecan repeat the procedure using a biased library containing insertscontaining part or all of the minimal linear portion plus one or moreadditional degenerate residues upstream or downstream thereof. Yeasttwo-hybrid screening methods also may be used to identify polypeptidesthat bind to the PARG polypeptides. Thus, the PARG polypeptides of theinvention, or a fragment thereof, can be used to screen peptidelibraries, including phage display libraries, to identify and selectpeptide binding partners of the PARG polypeptides of the invention. Suchmolecules can be used, as described, for screening assays, forpurification protocols, for interfering directly with the functioning ofPARG and for other purposes that will be apparent to those of ordinaryskill in the art.

A PARG polypeptide, or a fragment which contains the C-terminal PDZ4domain binding site, also can be used to isolate their native bindingpartners, including, e.g., the PTPL1 phosphatase that complexes withPARG. Isolation of phosphatases may be performed according to well-knownmethods. For example, isolated PARG polypeptides can be attached to asubstrate, and then a solution suspected of containing the phosphatasemay be applied to the substrate. If the phosphatase binding partner forPARG polypeptides is present in the solution, then it will bind to thesubstrate-bound PARG polypeptide. The phosphatase then may be isolated.Other proteins which are binding partners for PARG, such as otherproteins which contain PDZ4 domains may be isolated by similar methodswithout undue experimentation. Similarly, other proteins which bind PARG(e.g. Rho) can be isolated from biological samples and/or extracts bysuch methods.

Isolation of the PARG protein enables the skilled artisan to use theprotein for isolation of molecules which bind to it. For example,isolated PARG can be used to isolate PTPL1 and other proteins whichcontain PDZ4 domains. The PARG or PDZ binding fragment can beimmobilized on chromatographic media, such as polystyrene beads, or afilter, and the immobilized protein can be used to isolate proteinscontaining a PDZ4 domain from biological samples with no more thanroutine experimentation according to art-standard procedures foraffinity chromatography. Such procedures are described in greater detailbelow.

It will also be recognized that the invention embraces the use of thePARG cDNA sequences in expression vectors, as well as to transfect hostcells and cell lines, be these prokaryotic (e.g., E. coli), oreukaryotic (e.g., CHO cells, COS cells, yeast expression systems andrecombinant baculovirus expression in insect cells). Especially usefulare mammalian cells such as mouse, hamster, pig, goat, primate, etc.They may be of a wide variety of tissue types, and include primary cellsand cell lines. Specific examples include dendritic cells, U293 cells,peripheral blood leukocytes, bone marrow stem cells and embryonic stemcells. The expression vectors require that the pertinent sequence, i.e.,those nucleic acids described supra, be operably linked to a promoter.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712; incorporated by reference). Those ofskill in the art can readily determine the various parameters andconditions for producing antibody aerosols without resort to undueexperimentation. When using antisense preparations of the invention,slow intravenous administration is preferred.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, produces the desired response. Inthe case of treating cancer, the desired response is inhibiting theprogression of the cancer. This may involve only slowing the progressionof the disease temporarily, although more preferably, it involveshalting the progression of the disease permanently. This can bemonitored by routine methods or can be monitored according to diagnosticmethods of the invention discussed herein. Other therapeutic uses ofPARG include the modulation of actin reorganization, and modulation ofmast cell secretory granule release to treat allergic responses.

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. in vivo genetherapy using vectors such as adenovirus, retroviruses, herpes virus,and targeted liposomes also is contemplated according to the invention.

The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents active at the levelof a PARG or PARG fragment modulatable cellular function. In particular,such functions include Rho signal transduction and formation of aPTPL1-PARG protein complex. Generally, the screening methods involveassaying for compounds which interfere with a PARG activity such asPARG-PTPL1 binding, etc. Such methods are adaptable to automated, highthroughput screening of compounds. The target therapeutic indicationsfor pharmacological agents detected by the screening methods are limitedonly in that the target cellular function be subject to modulation byalteration of the formation of a complex comprising a PARG polypeptideor fragment thereof and one or more natural PARG intracellular bindingtargets, such as PTPL1 or other protein including a PDZ 4 domain. Targetindications include cellular processes modulated by Rho signaltransduction following receptor-ligand binding and PTPL1-mediatedphosphorylation.

A wide variety of assays for pharmacological agents are provided,including, labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays, cell-based assayssuch as two- or three-hybrid screens, expression assays, etc. Forexample, three-hybrid screens are used to rapidly examine the effect oftransfected nucleic acids on the intracellular binding of PARG or PARGfragments to specific intracellular targets. The transfected nucleicacids can encode, for example, combinatorial peptide libraries orantisense molecules. Convenient reagents for such assays, e.g., GAL4fusion proteins, are known in the art. An exemplary cell-based assayinvolves transfecting a cell with a nucleic acid encoding aPTPL1-binding PARG polypeptide (e.g., including a PDZ domain bindingsite) fused to a GAL4 DNA binding domain and a nucleic acid encoding aPTPL1 PDZ 4 domain fused to a transcription activation domain such asVP16. The cell also contains a reporter gene operably linked to a geneexpression regulatory region, such as one or more GAL4 binding sites.Activation of reporter gene transcription occurs when the PARG and PTPL1PDZ 4 fusion polypeptides bind such that the GAL4 DNA binding domain andthe VP 16 transcriptional activation domain are brought into proximityto enable transcription of the reporter gene. Agents which modulate aPARG polypeptide mediated cell function are then detected through achange in the expression of reporter gene. Methods for determiningchanges in the expression of a reporter gene are known in the art.

PARG fragments used in the methods, when not produced by a transfectednucleic acid are added to an assay mixture as an isolated polypeptide.PARG polypeptides preferably are produced recombinantly, although suchpolypeptides may be isolated from biological extracts. Recombinantlyproduced PARG polypeptides include chimeric proteins comprising a fusionof a PARG protein with another polypeptide, e.g., a polypeptide capableof providing or enhancing protein-protein binding, sequence specificnucleic acid binding (such as GAL4), enhancing stability of the PARGpolypeptide under assay conditions, or providing a detectable moiety,such as green fluorescent protein. A polypeptide fused to a PARGpolypeptide or fragment may also provide means of readily detecting thefusion protein, e.g., by immunological recognition or by fluorescentlabeling.

The assay mixture is comprised of a natural intracellular PARG bindingtarget such as a Rho protein, PTPL1 protein or fragment thereof capableof binding to PARG. While natural PARG binding targets may be used, itis frequently preferred to use portions (e.g., peptides or nucleic acidfragments) or analogs (i.e., agents which mimic the PARG bindingproperties of the natural binding target for purposes of the assay) ofthe PARG binding target so long as the portion or analog providesbinding affinity and avidity to the PARG fragment measurable in theassay.

The assay mixture also comprises a candidate pharmacological agent.Typically, a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a different response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateagents encompass numerous chemical classes, although typically they areorganic compounds. Preferably, the candidate pharmacological agents aresmall organic compounds, i.e., those having a molecular weight of morethan 50 yet less than about 2500, preferably less than about 1000 and,more preferably, less than about 500. Candidate agents comprisefunctional chemical groups necessary for structural interactions withpolypeptides and/or nucleic acids, and typically include at least anamine, carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups and more preferably at least three of thefunctional chemical groups. The candidate agents can comprise cycliccarbon or heterocyclic structure and/or aromatic or polyaromaticstructures substituted with one or more of the above-identifiedfunctional groups. Candidate agents also can be biomolecules such aspeptides, saccharides, fatty acids, sterols, isoprenoids, purines,pyrimidines, derivatives or structural analogs of the above, orcombinations thereof and the like. Where the agent is a nucleic acid,the agent typically is a DNA or RNA molecule, although modified nucleicacids as defined herein are also contemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease, inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The mixture of the foregoing assay materials is incubated underconditions whereby, but for the presence of the candidatepharmacological agent, the PARG polypeptide specifically binds thecellular binding target, a portion thereof or analog thereof. The orderof addition of components, incubation temperature, time of incubation,and other perimeters of the assay may be readily determined. Suchexperimentation merely involves optimization of the assay parameters,not the fundamental composition of the assay. Incubation temperaturestypically are between 4° C. and 40° C. Incubation times preferably areminimized to facilitate rapid, high throughput screening, and typicallyare between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe PARG polypeptide and one or more binding targets is detected by anyconvenient method available to the user. For cell free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components is immobilized on asolid substrate, from which the unbound components may be easilyseparated. The solid substrate can be made of a wide variety ofmaterials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximum signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromotograpic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. The transcript resulting from areporter gene transcription assay of PARG polypeptide binding to atarget molecule typically encodes a directly or indirectly detectableproduct, e.g., β-galactosidase activity, luciferase activity, and thelike. For cell free binding assays, one of the components usuallycomprises, or is coupled to, a detectable label. A wide variety oflabels can be used, such as those that provide direct detection (e.g.,radioactivity, luminescence, optical or electron density, etc). orindirect detection (e.g., epitope tag such as the FLAG epitope, enzymetag such as horseseradish peroxidase, etc.). The label may be bound to aPARG binding partner, or incorporated into the structure of the bindingpartner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,strepavidin-biotin conjugates, etc. Methods for detecting the labels arewell known in the art.

The invention provides PARG-specific binding agents, methods ofidentifying and making such agents, and their use in diagnosis, therapyand pharmaceutical development. For example, PARG-specificpharmacological agents are useful in a variety of diagnostic andtherapeutic applications, especially where disease or disease prognosisis associated with improper utilization of a pathway involving PARG,e.g., Rho activation, PTPL1-PARG complex formation, etc. NovelPARG-specific binding agents include PARG-specific antibodies and othernatural intracellular binding agents identified with assays such as twohybrid screens, and non-natural intracellular binding agents identifiedin screens of chemical libraries and the like.

In general, the specificity of PARG binding to a binding agent is shownby binding equilibrium constants. Targets which are capable ofselectively binding a PARG polypeptide preferably have bindingequilibrium constants of at least about 10⁷ M⁻¹, more preferably atleast about 10⁸ M⁻¹, and most preferably at least about 10⁹ M⁻¹. Thewide variety of cell based and cell free assays may be used todemonstrate PARG-specific binding. Cell based assays include one, twoand three hybrid screens, assays in which PARG-mediated transcription isinhibited or increased, etc. Cell free assays include PARG-proteinbinding assays, immunoassays, etc. Other assays useful for screeningagents which bind PARG polypeptides include fluorescence resonanceenergy transfer (FRET), and electrophoretic mobility shift analysis(EMSA).

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection with a retrovirus including thenucleic acid of interest, liposome mediated transfection, and the like.For certain uses, it is preferred to target the nucleic acid toparticular cells. In such instances, a vehicle used for delivering anucleic acid of the invention into a cell (e.g., a retrovirus, or othervirus; a liposome) can have a targeting molecule attached thereto. Forexample, a molecule such as an antibody specific for a surface membraneprotein on the target cell or a ligand for a receptor on the target cellcan be bound to or incorporated within the nucleic acid deliveryvehicle. For example, where liposomes are employed to deliver thenucleic acids of the invention, proteins which bind to a surfacemembrane protein associated with endocytosis may be incorporated intothe liposome formulation for targeting and/or to facilitate uptake. Suchproteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acids into cells, as is known by those skilled in the art. Suchsystems even permit oral delivery of nucleic acids.

EXAMPLES Example 1 Production of PDZ Fusion Proteins

To identify proteins that bind to the PDZ domains of PTPL1, regions ofPTPL1 cDNA corresponding to the various PDZ domains were produced bypolymerase chain reaction and subcloned into the GST fusion proteinexpression vector pGEX1λT (Pharmacia): GST-PDZ 1. amino acid residues1066-1166 of PTPL1; GST-PDZ 2-3. residues 1340-1579; GST-PDZ 3, residues1469-1579; GST-PDZ 4, residues 1762-1864; GST-PDZ 4-5, residues1762-1960 and GST-PDZ 5, residues 1856-1960 (FIG. 1A). Domains andmotifs indicated in FIG. 1A are: L, leucine zipper motif; Band 4.1, adomain of 300 amino acid residues with homology to the Band 4.1superfamily; P, PDZ domain; PTP, protein tyrosine phosphatase catalyticdomain; GST, glutathione S-transferase. The different expression vectorconstructs were transformed into E. coli. Glutathione S-transferase(GST) fusion proteins were produced and purified as described by Ridleyand Hall (Cell 70: 389-399, 1992) and then subjected to sodium dodecylsulfate (SDS)-gel electrophoresis. FIG. 1B shows that pure preparationsof fusion proteins with expected sizes were obtained.

Example 2 Identification of Proteins which Bind to PDZ4

PC-3 cells were obtained from American Type Culture Collection(Rockville, Md.) and cultured as described (Saras et al., 1994).Metabolic labeling of PC-3 cells was performed for 4 h in methionine-and cysteine-free MCDB 104 medium (Gibco/Life Technologies,Gaithersburg, Md.) with 150 Ci/ml of ³⁵S-methionine and ³⁵S-cysteine (invivo labeling mix; Amersham, Arlington Heights, Ill.). After labeling,the cells were solubilized in buffer containing 20 mM Tris-HCI, pH7.4,150 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate, 1 mMdithiothreitol, 1.5% Trasylol (Bayer, Germany) and 1 mMphenylmethylsulfonyl fluoride (Sigma, St. Louis, Mo.). After 15 min onice, cell debris was removed by centrifugation. Samples (1 ml) were thenincubated for 1.5 h at 4° C. with 10 μg of GST-PDZ fusion proteins boundto glutathione-Sepharose 4B beads (Pharmacia). The beads were pelletedand washed four times with solubilization buffer. The protein complexeswere eluted by boiling for 5 min in SDS-sample buffer (100 mM Tris-HCI,pH 8.8, 0.01% bromophenol blue, 36% glycerol, 4% SDS, 10 mMdithiothreitol) and analyzed by SDS-gel electrophoresis using 5-12%polyacrylamide gels (Blobel and Dobberstein, J. Cell Biol. 67: 835-851,1975). The gel was fixed, incubated with Amplify (Amersham) for 20 min,dried and subjected to fluorography. A component of 150 kDa that boundto the fusion proteins GST-PDZ 4 and GST-PDZ-4-5 could be observed (FIG.2); this component did not bind to GST fusion proteins containing PDZdomains 1, 2, 3 or 5 only, thus indicating that the 150 kDa componentinteracts specifically with PDZ 4 of PTPL1.

Example 3 Purification of 150 kDa Protein which Binds to PDZ4

In order to characterize the 150 kDa component further, it was purifiedfrom PC-3 cells. Briefly, immobilized fusion protein GST-PDZ 4 wasincubated with cell lysate from 1750 cm² of confluent PC-3 cellssolubilized as described above. Samples (20 ml) were incubated for 1.5 hat 4° C. with 200 μg of GST-PDZ 4 fusion protein bound-toglutathione-Sepharose 4B beads. The beads were washed and the boundproteins were eluted and subjected to SDS-get electrophoresis asdescribed above.

After staining of the gel with Coomassie Brilliant Blue, the band thatcontained the 150 kDa component was excised and subjected to in-geldigestion using modified trypsin or EndoLysC protease. The bandcontaining the 150 kDa component was transferred to Eppendorf tubes andsubjected to in-gel digestion (Hellman et al., Anal. Biochem. 224:451-455, 1995). In brief, the gel piece was washed with 0.2 M ammoniumbicarbonate (for digestion with trypsin) or 0.5 M Tris-HCl pH 9.2 (fordigestion with EndoLysC protease) and 50% acetonitrile, then driedcompletely. During rehydration, 0.5 μg of modified trypsin, sequencegrade (Promega, Madison, Wis.) or 0.5 μg of EndoLysC (WAKO Chemicals,Richmond, Va.) was added and 0.2 M ammonium bicarbonate (for trypsin) or0.1 M Tris-HCl pH 9.2 (for EndoLysC) was added in aliquots until the gelpiece was immersed. After overnight incubation at 30° C., thesupernatant was saved and combined with two further extractions from thegel piece. Generated peptides were isolated by reversed phase liquidchromatography using the SMART System (Pharmacia Biotech, Uppsala,Sweden). Peptides were sequenced on an Applied Biosystems (Foster City,Calif.) model 470A or 476A, following the manufacturers instructions.

Sequences were obtained from 10 peptides, and searches in differentdatabases showed that none of these sequences were found in any knowngene or protein, but the human Expressed Sequence Tags (ESTs) withGenBank accession numbers T32345, Z28937 and Z28520 (SEQ ID NOs:3, 4,5), contained cDNA sequences corresponding to three of the obtainedpeptides. Oligonucleotides corresponding to the nucleotide sequences ofthe ESTs were designed and used as probes for Northern blots andscreening of cDNA libraries.

Example 4 cDNA Cloning of PARG

The EST-derived oligonucleotides described above were used to screendifferent human cDNA libraries. Briefly, complementary and overlappingoligonucleotides corresponding to nucleotides 2-41 and 68-29 of an ESTwith the GenBank accession number Z28520 (SEQ ID NO:5) were made using aDNA synthesizer and labeled by a fill-in method (Sambrook et al., 1989)using the Klenow fragment of DNA polymerase I (Amersham) and α-³²P-dCTP(3000 Ci/mmol, Amersham). A λgt11 human skeletal muscle cDNA library(HL5002b; Clontech, Palo Alto, Calif.) was screened as described (Saraset al., 1994), using the ³²P-labeled oligonucleotides as a probe. Apositive clone was isolated, subcloned into pBluescript SK (Stratagene,La Jolla, Calif.) and thereafter sequenced.

Nucleotide sequencing revealed that the clone had a total length of 5237bp with an open reading frame of 3783 bp, coding for a protein of 1261amino acid residues. The open reading frame is flanked by a 5′untranslated sequence of 183 bp that contains an in frame stop codon atpositions 166-168, and a 3′ untranslated sequence of 1270 bp that has apoly(A) tail. The calculated molecular mass of the translated product is142 kDa and the protein was, for reasons described below, denoted PARG.The amino acid sequence of PARG (SEQ ID NO:2) is shown in FIG. 3A; thenucleotide sequence (SEQ ID NO:1) has been deposited in the EMBLdatabase.

Example 5 Structure of the PARG Protein

The amino acid sequence of PARG contained all peptide sequences obtainedpreviously (FIG. 3A). In the deduced amino acid sequence of PARG notransmembrane domain or signal sequence for secretion were found,indicating that PARG is likely an intracellular protein. Three regionswith homologies to other proteins could be identified: A GAP domain withsimilarity (23-33% amino acid sequence identity) to proteins of theRhoGAP family (Lamarche and Hall, 1994) is found at amino acid residues666-853, a cysteine-rich region at amino acid residues 613-652 hashomology to a regulatory, phorbol ester-, diacylglycerol- andZn2+-binding domain of members of the protein kinase C (PKC) family(Newton, 1995), and a region at amino acid residues 193-509 has homology(27% identity) to the gene product of the C. elegans gene ZK669.1 a(EMBL accession numberr Z37093). FIG. 3B shows an alignment of thelatter homology region, denoted ZPH region(for ZK667.1a-PARG homology).The alignment was done using the Clustal method (Higgins andSharp,CABIOS 5: 151-153, 1989), with some manual adjustment. Identicalamino acid residues are boxed. Like PARG, the gene product of ZK669.1 acontains in addition to the ZPH region, a cysteine-rich domain and a GAPdomain (FIG. 3C). Domains and motifs indicated in FIG. 3C are: ZPH,ZK669.1a-PARG Homology region; C, cysteine-rich domain; GAP, RhoGAPdomain.

Example 6 Expression of PARG mRNA

Northern blot analysis was performed to determine expression of the PARGmRNA. A Northern blot filter with mRNA from different human tissues waspurchased from Clontech. Each lane contained 2 μg of polyadenylated RNAfrom the indicated tissues. The filter was hybridized with the³²P-labeled oligonucleotide probe described above, at 42° C. overnightin a hybridization solution containing 50% formamide, 5×SSC (1×SSC is 15mM sodium citrate and 150 mM sodium chloride), 2×Denhardt's solution,0.5% SDS, 50 mM sodium phosphate, pH 6.9, and 0.1 mg/ml salmon spermDNA. The filter was washed two times in 0.5×SSC, 0.1% SDS at 55° C. for15 min. After washing, the filter was exposed to Amersham Hyperfilm MP.

Northern blot analysis of mRNA from various human tissues showed that asingle PARG transcript of 5.5 kb was found in all screened tissues (FIG.4). The expression of PARG mRNA was high in skeletal muscle and heartand moderate in placenta, liver and pancreas. Low expression wasobserved in brain, lung and kidney. The size of the transcript suggestedthat the cDNA clone obtained was close to full length.

Example 7 GAP Activity of PARG

In order to determine the GAP activity of PARG on proteins of the Rhofamily, the GAP domain of PARG was produced as a GST fusion protein inE. coli (FIG. 5A). Briefly, a DNA fragment coding for the GAP domain,i.e., amino acid residues 658-898, of PARG was produced by polymerasechain reaction and subcloned into pGEX1λT and referred to as GST-GAP.pGEX2T-based expression vectors containing RhoA, Rac1 and Cdc42 (G25Kisoform) cDNAs were obtained from Dr. A. Hall (MRC Laboratory forMolecular Cell Biology and Department of Biochemistry, UniversityCollege London, UK). These different expression vector constructs weretransformed into E. coli. The GST fusion proteins were produced andpurified essentially as described above in Example 1. Recombinant Rho,Rac and Cdc42 proteins were subjected to thrombin cleavage (Ridley andHall, 1992).

Recombinant Rho, Rac and Cdc42 were preloaded with γ-³²P-GTP andincubated for various time periods in the presence of the GST-GAP fusionprotein or, as control, GST protein. Thereafter, the radioactivity boundto the GTPase was determined as a measurement of the GTP hydrolysisactivity. Briefly, 200 nM aliquots of recombinant Rho, Rac and Cdc42were incubated at 30° C. with 10 μCi γ-³²P-GTP in 20 mM Tris-HCI, pH7.5, 25 mM NaCl, 4 mM EDTA, 0.1 mM dithiothreitol, and the nucleotideexchange was stopped after 10 min by the addition of 17 mM MgCl₂.Proteins (100 nM GST, 1 nM or 20 nM of GST-GAP fusion protein) wereadded to the reaction mixture and aliquots of 5 μl were withdrawn andcollected on nitrocellulose filters (HA, Millipore, Bedford, Mass.) at 3min intervals. The filters were washed with cold buffer (50 mM Tris-HCIpH 7.5, 50 mM NaCl, 5 mM MgCl₂), dried and subjected to scintillationcounting. The amount of protein-bound radioactivity is expressed as thepercentage of the total input.

The results show that the GAP domain of PARG, at the concentration of 1nM, had a strong GAP activity on Rho (FIG. 5B). At this concentration,no GAP activity on Rae or Cdc42 was detected (FIG. 5C and 5D). However,at a concentration of 20 nM, the GST-GAP fusion protein was also activeon Rae and Cdc42 (FIG. 5C and 5D). Thus, the results indicated that PARGhas a functional GAP domain which, in vitro, is active on Rho, Rae andCdc42, but with a clear preference for Rho. It is likely, therefore,that Rho is the physiological target of PARG. The name PARG isconsequently derived from PTPL1 Associated RhoGAP.

Example 8 PDZ4 Binds to the C-terminal Portion of PARG

It has been shown that PDZ domains interact with the C-terminal ends ofshort peptides and that a valine residue at the absolute C-terminal endis important for binding (Kim et al., 1995; Komau et al., 1995; Saras etal., in preparation). Since PARG was identified through a specificinteraction with PDZ 4 of PTPL1, and since it has a valine residue atthe C-terminal end, we found it likely that the interaction is mediatedvia PDZ 4 and the C-terminal tail of PARG. To verify this possibility,peptides corresponding to the last 4, 5 or 6 C-terminal amino acidresidues of PARG (PQFV, IPQFV and EIPQFV; SEQ ID Nos:7, 9 and 11) weresynthesized in an Applied Biosystems 430A Peptide Synthesizer usingt-butoxycarbonyl chemistry and purified by reversed phase highperformance liquid chromatography. The peptides were coupled to Affigel15 beads (Bio-Rad, Richmond, Calif.) via their N-terminal ends followingthe manufacturers instructions and incubated with GST-PDZ fusionproteins (50 nM) at 4° C. for 2 h in binding buffer (20 mM Tris-HCl, pH7.4, 150 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate, 1 mMdithiothreitol). The beads were washed four times in binding buffer andbound fusion proteins were eluted by boiling for 5 min in SDS-samplebuffer and subjected to SDS-gel electrophoresis using 11% polyacrylamidegels. After electrbphoresis, the proteins were transferred tonitrocellulose membranes (Hybond C Extra; Amersham) and the membraneswere incubated with α-GST antiserum (rabbit antiserum raised againstrecombinant GST expressed in bacteria). Bound antibodies were visualizedby using enhanced chemiluminescence (ECL, Amersham), according to themanufacturer's instructions.

As shown in FIG. 6, the fusion proteins GST-PDZ 4 and GST-PDZ 4-5, butnot GST fusion proteins containing PDZ 1, PDZ 2, PDZ 3 or PDZ 5 only,bound to the peptide corresponding to the last four amino acid residuesof PARG. Similar results were obtained by using the longer peptides,indicating that a maximum of four amino acid residues at the C-terminalend of PARG is enough for a strong and specific interaction with PDZ 4of PTPL1.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

A sequence listing is presented below and is followed by what isclaimed.

39 1 5238 DNA Homo sapiens CDS 184..3966 1 gctgtggctg cggctgcggctgcggctgag atttggccgg gcgtccgcag gccgtggggg 60 atgggggcag cgagctccagccctcggcgg tggcggcggc cgtaggtgtg gggcgggcgt 120 ccgcgtccgg cacgcgagatggagcgccgt ggatttcagt ttttctgact gttacatgaa 180 agg atg att gct cac aaacag aaa aag aca aag aaa aaa cgt gct tgg 228 Met Ile Ala His Lys Gln LysLys Thr Lys Lys Lys Arg Ala Trp 1 5 10 15 gca tca ggt caa ctc tct actgat att aca act tct gaa atg ggg ctc 276 Ala Ser Gly Gln Leu Ser Thr AspIle Thr Thr Ser Glu Met Gly Leu 20 25 30 aag tcc tta agt tcc aac tct attttt gat ccg gat tac atc aag gag 324 Lys Ser Leu Ser Ser Asn Ser Ile PheAsp Pro Asp Tyr Ile Lys Glu 35 40 45 ttg gtg aat gat atc agg aag ttc tcccac atc tta cta tat ttg aaa 372 Leu Val Asn Asp Ile Arg Lys Phe Ser HisIle Leu Leu Tyr Leu Lys 50 55 60 gaa gcc ata ttt tca gac tgt ttt aaa gaagtt att cat ata cgt cta 420 Glu Ala Ile Phe Ser Asp Cys Phe Lys Glu ValIle His Ile Arg Leu 65 70 75 gag gaa ctg ctc cgt gtt tta aag tct ata atgaat aaa cat cag aac 468 Glu Glu Leu Leu Arg Val Leu Lys Ser Ile Met AsnLys His Gln Asn 80 85 90 95 ctc aat tct gtt gat ctt caa aat gct gca gaaatg ctc act gca aaa 516 Leu Asn Ser Val Asp Leu Gln Asn Ala Ala Glu MetLeu Thr Ala Lys 100 105 110 gtg aaa gct gtg aac ttc aca gaa gtt aat gaagaa aac aaa aac gat 564 Val Lys Ala Val Asn Phe Thr Glu Val Asn Glu GluAsn Lys Asn Asp 115 120 125 ctc ttc cag gaa gtg ttt tct tct att gaa actttg gca ttt acc ttt 612 Leu Phe Gln Glu Val Phe Ser Ser Ile Glu Thr LeuAla Phe Thr Phe 130 135 140 gga aat atc ctt aca aac ttc ctt atg gga gatgta ggc aat gat tca 660 Gly Asn Ile Leu Thr Asn Phe Leu Met Gly Asp ValGly Asn Asp Ser 145 150 155 ttc ttg cga ctg cct gtt tct cga gaa act aagtcg ttt gaa aat gtt 708 Phe Leu Arg Leu Pro Val Ser Arg Glu Thr Lys SerPhe Glu Asn Val 160 165 170 175 tct gtg gaa tca gtg gac tca tcc agt gaaaaa gga aat ttt tcc cct 756 Ser Val Glu Ser Val Asp Ser Ser Ser Glu LysGly Asn Phe Ser Pro 180 185 190 tta gaa cta gac aac gtg ctg tta aag aacact gac tct atc gag ctg 804 Leu Glu Leu Asp Asn Val Leu Leu Lys Asn ThrAsp Ser Ile Glu Leu 195 200 205 gct ttg tca tat gct aaa act tgg tca aaatat act aag aac ata gtt 852 Ala Leu Ser Tyr Ala Lys Thr Trp Ser Lys TyrThr Lys Asn Ile Val 210 215 220 tca tgg gtt gaa aaa aag ctt aac ttg gaattg gag tcc act aga aat 900 Ser Trp Val Glu Lys Lys Leu Asn Leu Glu LeuGlu Ser Thr Arg Asn 225 230 235 atg gtc aag ttg gca gag gca act aga actaac att gga att cag gag 948 Met Val Lys Leu Ala Glu Ala Thr Arg Thr AsnIle Gly Ile Gln Glu 240 245 250 255 ttc atg cca ctg cag tct ctg ttt actaat gct ctt ctt aat gat ata 996 Phe Met Pro Leu Gln Ser Leu Phe Thr AsnAla Leu Leu Asn Asp Ile 260 265 270 gaa agc agt cac ctt tta caa caa acaatt gca gct ctc cag gct aac 1044 Glu Ser Ser His Leu Leu Gln Gln Thr IleAla Ala Leu Gln Ala Asn 275 280 285 aaa ttt gtg cag cct cta ctt gga aggaaa aat gaa atg gaa aaa caa 1092 Lys Phe Val Gln Pro Leu Leu Gly Arg LysAsn Glu Met Glu Lys Gln 290 295 300 agg aaa gaa ata aaa gag ctt tgg aaacag gag caa aat aaa atg ctt 1140 Arg Lys Glu Ile Lys Glu Leu Trp Lys GlnGlu Gln Asn Lys Met Leu 305 310 315 gaa gca gag aat gct ctc aaa aag gcaaaa tta tta tgc atg caa cgt 1188 Glu Ala Glu Asn Ala Leu Lys Lys Ala LysLeu Leu Cys Met Gln Arg 320 325 330 335 caa gat gaa tat gag aaa gca aagtct tcc atg ttt cgt gca gaa gag 1236 Gln Asp Glu Tyr Glu Lys Ala Lys SerSer Met Phe Arg Ala Glu Glu 340 345 350 gag cat ctg tct tca agt ggc ggatta gca aaa aat ctc aac aag caa 1284 Glu His Leu Ser Ser Ser Gly Gly LeuAla Lys Asn Leu Asn Lys Gln 355 360 365 cta gaa aaa aag cga agg ttg gaagag gag gct ctc caa aaa gta gaa 1332 Leu Glu Lys Lys Arg Arg Leu Glu GluGlu Ala Leu Gln Lys Val Glu 370 375 380 gaa gca gat gaa ctt tac aaa gtttgt gtg aca aat gtt gaa gaa aga 1380 Glu Ala Asp Glu Leu Tyr Lys Val CysVal Thr Asn Val Glu Glu Arg 385 390 395 aga aat gat gta gaa aat acc aaaaga gaa att tta gca caa ctc cgg 1428 Arg Asn Asp Val Glu Asn Thr Lys ArgGlu Ile Leu Ala Gln Leu Arg 400 405 410 415 aca ctt gtt ttc cag tgt gatctt acc ctt aaa gcg gta aca gtt aac 1476 Thr Leu Val Phe Gln Cys Asp LeuThr Leu Lys Ala Val Thr Val Asn 420 425 430 ctc ttc cac atg cag cat ctgcag gct gct tcc ctt gca gac aga tta 1524 Leu Phe His Met Gln His Leu GlnAla Ala Ser Leu Ala Asp Arg Leu 435 440 445 cag tct ctc tgt ggt agt gccaaa ctc tat gac cca ggc caa gag tac 1572 Gln Ser Leu Cys Gly Ser Ala LysLeu Tyr Asp Pro Gly Gln Glu Tyr 450 455 460 agt gaa ttt gtc aag gcc acaaat tca act gaa gaa gaa aaa gtt gat 1620 Ser Glu Phe Val Lys Ala Thr AsnSer Thr Glu Glu Glu Lys Val Asp 465 470 475 gga aat gta aat aaa cat ttaaat agt tcc caa cct tca gga ttt gga 1668 Gly Asn Val Asn Lys His Leu AsnSer Ser Gln Pro Ser Gly Phe Gly 480 485 490 495 cct gcc aac tct tta gaggat gtt gta cgc ctt cct gac agt tct aat 1716 Pro Ala Asn Ser Leu Glu AspVal Val Arg Leu Pro Asp Ser Ser Asn 500 505 510 aaa att gaa gag gac agatgc tct aac agt gca gat ata aca ggt cct 1764 Lys Ile Glu Glu Asp Arg CysSer Asn Ser Ala Asp Ile Thr Gly Pro 515 520 525 tcc ttt ata aga tca tggaca ttt ggg atg ttt agt gat tct gag agc 1812 Ser Phe Ile Arg Ser Trp ThrPhe Gly Met Phe Ser Asp Ser Glu Ser 530 535 540 act gga ggg agc agc gaatct aga tct ctg gat tca gaa tct ata agt 1860 Thr Gly Gly Ser Ser Glu SerArg Ser Leu Asp Ser Glu Ser Ile Ser 545 550 555 cca gga gac ttt cat cgaaaa ctt cca cga aca cca tcc agt gga act 1908 Pro Gly Asp Phe His Arg LysLeu Pro Arg Thr Pro Ser Ser Gly Thr 560 565 570 575 atg tcc tct gca gatgat cta gat gaa aga gag cca cct tcc cct tca 1956 Met Ser Ser Ala Asp AspLeu Asp Glu Arg Glu Pro Pro Ser Pro Ser 580 585 590 gaa act gga ccc aattcc ctt gga aca ttt aag aaa aca ttg atg tca 2004 Glu Thr Gly Pro Asn SerLeu Gly Thr Phe Lys Lys Thr Leu Met Ser 595 600 605 aag gca gct ctc acacac aag ttt cgc aaa ttg aga tcc ccc acg aaa 2052 Lys Ala Ala Leu Thr HisLys Phe Arg Lys Leu Arg Ser Pro Thr Lys 610 615 620 tgt agg gat tgt gaaggc att gta gtg ttc caa ggt gtt gaa tgt gaa 2100 Cys Arg Asp Cys Glu GlyIle Val Val Phe Gln Gly Val Glu Cys Glu 625 630 635 gag tgt ctc ctt gtttgt cat cga aag tgt ttg gaa aat tta gtc att 2148 Glu Cys Leu Leu Val CysHis Arg Lys Cys Leu Glu Asn Leu Val Ile 640 645 650 655 att tgt ggt catcag aaa ctt cca gga aaa ata cac tta ttt gga gca 2196 Ile Cys Gly His GlnLys Leu Pro Gly Lys Ile His Leu Phe Gly Ala 660 665 670 gaa ttc aca ctagtt gca aaa aag gaa cca gat ggt atc cct ttt ata 2244 Glu Phe Thr Leu ValAla Lys Lys Glu Pro Asp Gly Ile Pro Phe Ile 675 680 685 ctc aaa ata tgtgcc tca gag att gaa aat aga gct ttg tgt cta cag 2292 Leu Lys Ile Cys AlaSer Glu Ile Glu Asn Arg Ala Leu Cys Leu Gln 690 695 700 gga att tat cgtgtg tgt gga aac aaa ata aaa act gaa aaa ttg tgt 2340 Gly Ile Tyr Arg ValCys Gly Asn Lys Ile Lys Thr Glu Lys Leu Cys 705 710 715 cta gct ttg gaaaat ggt atg cac ttg gta gat att tca gaa ttt agt 2388 Leu Ala Leu Glu AsnGly Met His Leu Val Asp Ile Ser Glu Phe Ser 720 725 730 735 tca cat gatatc tgt gac gtc ttg aaa tta tac ctt cgg cag ctc cca 2436 Ser His Asp IleCys Asp Val Leu Lys Leu Tyr Leu Arg Gln Leu Pro 740 745 750 gaa cca tttatt tta ttt cga ttg tac aag gaa ttt ata gac ctt gca 2484 Glu Pro Phe IleLeu Phe Arg Leu Tyr Lys Glu Phe Ile Asp Leu Ala 755 760 765 aaa gag atccaa cat gta aat gaa gaa caa gag aca aaa aag aat agt 2532 Lys Glu Ile GlnHis Val Asn Glu Glu Gln Glu Thr Lys Lys Asn Ser 770 775 780 ctt gaa gacaaa aaa tgg cca aat atg tgt ata gaa ata aac cga att 2580 Leu Glu Asp LysLys Trp Pro Asn Met Cys Ile Glu Ile Asn Arg Ile 785 790 795 ctt cta aaaagc aaa gac ctt cta aga caa ttg cca gca tca aat ttt 2628 Leu Leu Lys SerLys Asp Leu Leu Arg Gln Leu Pro Ala Ser Asn Phe 800 805 810 815 aac agtctt cat ttc ctt ata gta cat cta aag cgg gta gta gat cat 2676 Asn Ser LeuHis Phe Leu Ile Val His Leu Lys Arg Val Val Asp His 820 825 830 gca gaagaa aac aag atg aac tcc aaa aac ttg ggg gtg ata ttt gga 2724 Ala Glu GluAsn Lys Met Asn Ser Lys Asn Leu Gly Val Ile Phe Gly 835 840 845 cca agtctc att agg cca agg cca caa act gct cct atc acc atc tcc 2772 Pro Ser LeuIle Arg Pro Arg Pro Gln Thr Ala Pro Ile Thr Ile Ser 850 855 860 tcc cttgca gag tat tca aat caa gca cgc ttg gta gag ttt ctc att 2820 Ser Leu AlaGlu Tyr Ser Asn Gln Ala Arg Leu Val Glu Phe Leu Ile 865 870 875 act tactca cag aag atc ttc gat ggg tcc cta caa cca caa gat gtt 2868 Thr Tyr SerGln Lys Ile Phe Asp Gly Ser Leu Gln Pro Gln Asp Val 880 885 890 895 atgtgt agc ata ggt gtt gtt gat caa ggc tgt ttt cca aag cct ctg 2916 Met CysSer Ile Gly Val Val Asp Gln Gly Cys Phe Pro Lys Pro Leu 900 905 910 ttatca cca gaa gaa aga gac att gaa cgt tcc atg aag tca cta ttt 2964 Leu SerPro Glu Glu Arg Asp Ile Glu Arg Ser Met Lys Ser Leu Phe 915 920 925 ttttct tca aag gaa gat atc cat act tca gag agt gaa agc aaa att 3012 Phe SerSer Lys Glu Asp Ile His Thr Ser Glu Ser Glu Ser Lys Ile 930 935 940 tttgaa cga gct aca tca ttt gag gaa tca gaa cgc aag caa aat gcg 3060 Phe GluArg Ala Thr Ser Phe Glu Glu Ser Glu Arg Lys Gln Asn Ala 945 950 955 ttagga aaa tgt gat gca tgt ctc agt gac aaa gca cag ttg ctt cta 3108 Leu GlyLys Cys Asp Ala Cys Leu Ser Asp Lys Ala Gln Leu Leu Leu 960 965 970 975gac caa gag gct gaa tca gca tcc caa aag ata gaa gat ggt aaa gcc 3156 AspGln Glu Ala Glu Ser Ala Ser Gln Lys Ile Glu Asp Gly Lys Ala 980 985 990cct aag cca ctt tct ctg aaa tct gat agg tca aca aac aat gtg gag 3204 ProLys Pro Leu Ser Leu Lys Ser Asp Arg Ser Thr Asn Asn Val Glu 995 10001005 agg cat act cca agg acc aag att aga cct gta agt ttg cct gta gat3252 Arg His Thr Pro Arg Thr Lys Ile Arg Pro Val Ser Leu Pro Val Asp1010 1015 1020 aga cta ctt ctt gca agt cct cct aat gag aga aat ggc agaaat atg 3300 Arg Leu Leu Leu Ala Ser Pro Pro Asn Glu Arg Asn Gly Arg AsnMet 1025 1030 1035 gga aat gta aat tta gac aag ttt tgc aag aat cct gccttt gaa gga 3348 Gly Asn Val Asn Leu Asp Lys Phe Cys Lys Asn Pro Ala PheGlu Gly 1040 1045 1050 1055 gtt aat aga aaa gac gct gct act act gtt tgttcc aaa ttt aat ggc 3396 Val Asn Arg Lys Asp Ala Ala Thr Thr Val Cys SerLys Phe Asn Gly 1060 1065 1070 ttt gac cag caa act cta cag aaa att caggac aaa cag tat gaa caa 3444 Phe Asp Gln Gln Thr Leu Gln Lys Ile Gln AspLys Gln Tyr Glu Gln 1075 1080 1085 aac agc cta act gcc aag act aca atgatc atg ccc agt gca ctc cag 3492 Asn Ser Leu Thr Ala Lys Thr Thr Met IleMet Pro Ser Ala Leu Gln 1090 1095 1100 gaa aaa gga gtg aca aca agc ctccag att agt ggg gac cat tct atc 3540 Glu Lys Gly Val Thr Thr Ser Leu GlnIle Ser Gly Asp His Ser Ile 1105 1110 1115 aat gcc act caa ccc agt aagcca tat gca gag cca gtc agg tca gtg 3588 Asn Ala Thr Gln Pro Ser Lys ProTyr Ala Glu Pro Val Arg Ser Val 1120 1125 1130 1135 aga gag gca tct gagaga cgg tct tca gat tcc tac cct ctc gct cct 3636 Arg Glu Ala Ser Glu ArgArg Ser Ser Asp Ser Tyr Pro Leu Ala Pro 1140 1145 1150 gtc aga gca cccaga aca ctg cag cct caa cat tgg aca aca ttt tat 3684 Val Arg Ala Pro ArgThr Leu Gln Pro Gln His Trp Thr Thr Phe Tyr 1155 1160 1165 aaa cca catgct ccc atc atc agt atc agg ggg aat gag gag aag cca 3732 Lys Pro His AlaPro Ile Ile Ser Ile Arg Gly Asn Glu Glu Lys Pro 1170 1175 1180 gct tcaccc tca gca gca tgc cct cct ggc aca gat cac gat ccc cac 3780 Ala Ser ProSer Ala Ala Cys Pro Pro Gly Thr Asp His Asp Pro His 1185 1190 1195 ggtctc gtg gtg aag tca atg cca gac cca gac aaa gca tca gct tgt 3828 Gly LeuVal Val Lys Ser Met Pro Asp Pro Asp Lys Ala Ser Ala Cys 1200 1205 12101215 cct ggg caa gca act ggt caa cct aaa gaa gac tct gag gag ctt ggc3876 Pro Gly Gln Ala Thr Gly Gln Pro Lys Glu Asp Ser Glu Glu Leu Gly1220 1225 1230 ttg cct gat gtg aat cca atg tgt cag aga cca agg cta aaacga atg 3924 Leu Pro Asp Val Asn Pro Met Cys Gln Arg Pro Arg Leu Lys ArgMet 1235 1240 1245 caa cag ttt gaa gac ctc gaa gat gaa att cca caa tttgtg 3966 Gln Gln Phe Glu Asp Leu Glu Asp Glu Ile Pro Gln Phe Val 12501255 1260 tagggatgtc aaatttcagg gtttttttgt tgttgttgtg ttattttgtggtattgtgct 4026 tgttttgtga aagaatgttt tgacagggcc ccttttgtat aggactgccaaatcatgggt 4086 tttgcctttt gttgttgtat ttatcctctg ttggtaatac tgaatggtagaatgttttga 4146 tagggtcaca tttgtgcctc actggaatta tctttaaatt ctgtatttttaaagttgtga 4206 ataagatagg tggattcgta ttttttaaag ttcagttgac tttccccaccaaatggtcca 4266 tttgaatgca tccctaatat atgatatagt ctcaactaat aggtgcaatttgggaaaatc 4326 aggtttattt tttggagtgg aactgttata agtgcttatt tataaaaggaatgtttctga 4386 atgcaagtgc ctaaaaagat ctttgttggt atgcatatgt tttgtcacacaatttatagt 4446 gcatctttca ccatttgtgc ttttttaaga tagtatgtaa gctcttatttttcaattggc 4506 aattcagtta atttttaaat gtttacataa tggccagaag gcttgcaaatctgtatttaa 4566 ttgcatttta attaattgcc agtttttaca tgtagtagtc agttgtacaaagaaaatgca 4626 cttaaacctg tttctaaatt atatattcag ttatattata tttggctttagatggtttta 4686 atacatttga tagtttttca ccccttggct ttattttata taaacttttgtttttcagca 4746 gttctgaact ttttagtatt ttataaatgg tccaaaaaat gcctgtttcagaagtttttg 4806 aattcagtgc atttcctctt gatttgtctg ggttaaaacc attccttttgtatgaaatgt 4866 tttgacttag gaatcatttt atgtacttgt tctacctgga ttgtcaacaactgaaagtac 4926 atatttcatc caaatcaagc taaaatttat ttaagttgat tctgagagtacaggtcagta 4986 agcctcatta tttggaattt gagagaagta taggtgatcg gatctgtttcatttataaaa 5046 ggtccagttt ttaggactag tacattcctg ttattttctg ggttttatcattttgcctaa 5106 aataggatat aaaagggaca aaaaataagt agactgtttt tatgtgtgaattatatttct 5166 actaaatgtt tttgtatgac tgtgttatac ttgataatat atatatatatatataaaaaa 5226 aaaaaaaaaa aa 5238 2 1261 PRT Homo sapiens 2 Met Ile AlaHis Lys Gln Lys Lys Thr Lys Lys Lys Arg Ala Trp Ala 1 5 10 15 Ser GlyGln Leu Ser Thr Asp Ile Thr Thr Ser Glu Met Gly Leu Lys 20 25 30 Ser LeuSer Ser Asn Ser Ile Phe Asp Pro Asp Tyr Ile Lys Glu Leu 35 40 45 Val AsnAsp Ile Arg Lys Phe Ser His Ile Leu Leu Tyr Leu Lys Glu 50 55 60 Ala IlePhe Ser Asp Cys Phe Lys Glu Val Ile His Ile Arg Leu Glu 65 70 75 80 GluLeu Leu Arg Val Leu Lys Ser Ile Met Asn Lys His Gln Asn Leu 85 90 95 AsnSer Val Asp Leu Gln Asn Ala Ala Glu Met Leu Thr Ala Lys Val 100 105 110Lys Ala Val Asn Phe Thr Glu Val Asn Glu Glu Asn Lys Asn Asp Leu 115 120125 Phe Gln Glu Val Phe Ser Ser Ile Glu Thr Leu Ala Phe Thr Phe Gly 130135 140 Asn Ile Leu Thr Asn Phe Leu Met Gly Asp Val Gly Asn Asp Ser Phe145 150 155 160 Leu Arg Leu Pro Val Ser Arg Glu Thr Lys Ser Phe Glu AsnVal Ser 165 170 175 Val Glu Ser Val Asp Ser Ser Ser Glu Lys Gly Asn PheSer Pro Leu 180 185 190 Glu Leu Asp Asn Val Leu Leu Lys Asn Thr Asp SerIle Glu Leu Ala 195 200 205 Leu Ser Tyr Ala Lys Thr Trp Ser Lys Tyr ThrLys Asn Ile Val Ser 210 215 220 Trp Val Glu Lys Lys Leu Asn Leu Glu LeuGlu Ser Thr Arg Asn Met 225 230 235 240 Val Lys Leu Ala Glu Ala Thr ArgThr Asn Ile Gly Ile Gln Glu Phe 245 250 255 Met Pro Leu Gln Ser Leu PheThr Asn Ala Leu Leu Asn Asp Ile Glu 260 265 270 Ser Ser His Leu Leu GlnGln Thr Ile Ala Ala Leu Gln Ala Asn Lys 275 280 285 Phe Val Gln Pro LeuLeu Gly Arg Lys Asn Glu Met Glu Lys Gln Arg 290 295 300 Lys Glu Ile LysGlu Leu Trp Lys Gln Glu Gln Asn Lys Met Leu Glu 305 310 315 320 Ala GluAsn Ala Leu Lys Lys Ala Lys Leu Leu Cys Met Gln Arg Gln 325 330 335 AspGlu Tyr Glu Lys Ala Lys Ser Ser Met Phe Arg Ala Glu Glu Glu 340 345 350His Leu Ser Ser Ser Gly Gly Leu Ala Lys Asn Leu Asn Lys Gln Leu 355 360365 Glu Lys Lys Arg Arg Leu Glu Glu Glu Ala Leu Gln Lys Val Glu Glu 370375 380 Ala Asp Glu Leu Tyr Lys Val Cys Val Thr Asn Val Glu Glu Arg Arg385 390 395 400 Asn Asp Val Glu Asn Thr Lys Arg Glu Ile Leu Ala Gln LeuArg Thr 405 410 415 Leu Val Phe Gln Cys Asp Leu Thr Leu Lys Ala Val ThrVal Asn Leu 420 425 430 Phe His Met Gln His Leu Gln Ala Ala Ser Leu AlaAsp Arg Leu Gln 435 440 445 Ser Leu Cys Gly Ser Ala Lys Leu Tyr Asp ProGly Gln Glu Tyr Ser 450 455 460 Glu Phe Val Lys Ala Thr Asn Ser Thr GluGlu Glu Lys Val Asp Gly 465 470 475 480 Asn Val Asn Lys His Leu Asn SerSer Gln Pro Ser Gly Phe Gly Pro 485 490 495 Ala Asn Ser Leu Glu Asp ValVal Arg Leu Pro Asp Ser Ser Asn Lys 500 505 510 Ile Glu Glu Asp Arg CysSer Asn Ser Ala Asp Ile Thr Gly Pro Ser 515 520 525 Phe Ile Arg Ser TrpThr Phe Gly Met Phe Ser Asp Ser Glu Ser Thr 530 535 540 Gly Gly Ser SerGlu Ser Arg Ser Leu Asp Ser Glu Ser Ile Ser Pro 545 550 555 560 Gly AspPhe His Arg Lys Leu Pro Arg Thr Pro Ser Ser Gly Thr Met 565 570 575 SerSer Ala Asp Asp Leu Asp Glu Arg Glu Pro Pro Ser Pro Ser Glu 580 585 590Thr Gly Pro Asn Ser Leu Gly Thr Phe Lys Lys Thr Leu Met Ser Lys 595 600605 Ala Ala Leu Thr His Lys Phe Arg Lys Leu Arg Ser Pro Thr Lys Cys 610615 620 Arg Asp Cys Glu Gly Ile Val Val Phe Gln Gly Val Glu Cys Glu Glu625 630 635 640 Cys Leu Leu Val Cys His Arg Lys Cys Leu Glu Asn Leu ValIle Ile 645 650 655 Cys Gly His Gln Lys Leu Pro Gly Lys Ile His Leu PheGly Ala Glu 660 665 670 Phe Thr Leu Val Ala Lys Lys Glu Pro Asp Gly IlePro Phe Ile Leu 675 680 685 Lys Ile Cys Ala Ser Glu Ile Glu Asn Arg AlaLeu Cys Leu Gln Gly 690 695 700 Ile Tyr Arg Val Cys Gly Asn Lys Ile LysThr Glu Lys Leu Cys Leu 705 710 715 720 Ala Leu Glu Asn Gly Met His LeuVal Asp Ile Ser Glu Phe Ser Ser 725 730 735 His Asp Ile Cys Asp Val LeuLys Leu Tyr Leu Arg Gln Leu Pro Glu 740 745 750 Pro Phe Ile Leu Phe ArgLeu Tyr Lys Glu Phe Ile Asp Leu Ala Lys 755 760 765 Glu Ile Gln His ValAsn Glu Glu Gln Glu Thr Lys Lys Asn Ser Leu 770 775 780 Glu Asp Lys LysTrp Pro Asn Met Cys Ile Glu Ile Asn Arg Ile Leu 785 790 795 800 Leu LysSer Lys Asp Leu Leu Arg Gln Leu Pro Ala Ser Asn Phe Asn 805 810 815 SerLeu His Phe Leu Ile Val His Leu Lys Arg Val Val Asp His Ala 820 825 830Glu Glu Asn Lys Met Asn Ser Lys Asn Leu Gly Val Ile Phe Gly Pro 835 840845 Ser Leu Ile Arg Pro Arg Pro Gln Thr Ala Pro Ile Thr Ile Ser Ser 850855 860 Leu Ala Glu Tyr Ser Asn Gln Ala Arg Leu Val Glu Phe Leu Ile Thr865 870 875 880 Tyr Ser Gln Lys Ile Phe Asp Gly Ser Leu Gln Pro Gln AspVal Met 885 890 895 Cys Ser Ile Gly Val Val Asp Gln Gly Cys Phe Pro LysPro Leu Leu 900 905 910 Ser Pro Glu Glu Arg Asp Ile Glu Arg Ser Met LysSer Leu Phe Phe 915 920 925 Ser Ser Lys Glu Asp Ile His Thr Ser Glu SerGlu Ser Lys Ile Phe 930 935 940 Glu Arg Ala Thr Ser Phe Glu Glu Ser GluArg Lys Gln Asn Ala Leu 945 950 955 960 Gly Lys Cys Asp Ala Cys Leu SerAsp Lys Ala Gln Leu Leu Leu Asp 965 970 975 Gln Glu Ala Glu Ser Ala SerGln Lys Ile Glu Asp Gly Lys Ala Pro 980 985 990 Lys Pro Leu Ser Leu LysSer Asp Arg Ser Thr Asn Asn Val Glu Arg 995 1000 1005 His Thr Pro ArgThr Lys Ile Arg Pro Val Ser Leu Pro Val Asp Arg 1010 1015 1020 Leu LeuLeu Ala Ser Pro Pro Asn Glu Arg Asn Gly Arg Asn Met Gly 1025 1030 10351040 Asn Val Asn Leu Asp Lys Phe Cys Lys Asn Pro Ala Phe Glu Gly Val1045 1050 1055 Asn Arg Lys Asp Ala Ala Thr Thr Val Cys Ser Lys Phe AsnGly Phe 1060 1065 1070 Asp Gln Gln Thr Leu Gln Lys Ile Gln Asp Lys GlnTyr Glu Gln Asn 1075 1080 1085 Ser Leu Thr Ala Lys Thr Thr Met Ile MetPro Ser Ala Leu Gln Glu 1090 1095 1100 Lys Gly Val Thr Thr Ser Leu GlnIle Ser Gly Asp His Ser Ile Asn 1105 1110 1115 1120 Ala Thr Gln Pro SerLys Pro Tyr Ala Glu Pro Val Arg Ser Val Arg 1125 1130 1135 Glu Ala SerGlu Arg Arg Ser Ser Asp Ser Tyr Pro Leu Ala Pro Val 1140 1145 1150 ArgAla Pro Arg Thr Leu Gln Pro Gln His Trp Thr Thr Phe Tyr Lys 1155 11601165 Pro His Ala Pro Ile Ile Ser Ile Arg Gly Asn Glu Glu Lys Pro Ala1170 1175 1180 Ser Pro Ser Ala Ala Cys Pro Pro Gly Thr Asp His Asp ProHis Gly 1185 1190 1195 1200 Leu Val Val Lys Ser Met Pro Asp Pro Asp LysAla Ser Ala Cys Pro 1205 1210 1215 Gly Gln Ala Thr Gly Gln Pro Lys GluAsp Ser Glu Glu Leu Gly Leu 1220 1225 1230 Pro Asp Val Asn Pro Met CysGln Arg Pro Arg Leu Lys Arg Met Gln 1235 1240 1245 Gln Phe Glu Asp LeuGlu Asp Glu Ile Pro Gln Phe Val 1250 1255 1260 3 251 DNA Homo sapiensunsure 201..201 n = a, c, g or t 3 ttaatagaaa agacgctgct actactgtttgttccaaatt taatggcttt gaccagcaaa 60 ctctacagaa aattcaggac aaacagtatgaacaaaacag cctaactgcc aagactacaa 120 tgatcatgcc cagtgcactc caggaaaaaggagtgacaac aagcctccag attagtgggg 180 accattctat caatgccact naacccagtaagccatatgc agagccagtc aggtcagtga 240 gagaggcatc t 251 4 256 DNA Homosapiens unsure 36..36 n = a, c, g or t 4 cggtaagcca agctcctcagagtcttcttt aggttnacca gttgcttgcc caggacaagc 60 tgatgctttg tctgggtctggcattgactt caccacgaga ccgtggggat cgtgatctgt 120 gccaggaggc actgctgctgagggtgaagc tggcttctcc tcattccccc tgatactgat 180 gatgggagca tgtggtttataaaatgttgt ccaatgttga ggctgcagtg ttctgggtgc 240 tctgacagga gcgaga 256 5298 DNA Homo sapiens unsure 140..140 n = a, c, g or t 5 ctttctgtgatagtgccaaa ctctatgacc caggccaaga gtacagtgaa tttgtcaagg 60 ccacaaattcaactgaagaa gaaaaagttg atggaaatgt aaataaacat ttaaatagtt 120 cccaaccttcaggatttggn cctgccaact ctttagagga tgttgtacgc cttcctgaca 180 gttctaataaaattgaagag gacagatgct ctaacagtgc agntataaca ggtccttcct 240 ttataagatcatggacattt gggatgttta gtgattctga gagcactgga gggagcag 298 6 12 DNA Homosapiens 6 ccacaatttg tg 12 7 4 PRT Homo sapiens 7 Pro Gln Phe Val 1 8 15DNA Homo sapiens 8 attccacaat ttgtg 15 9 5 PRT Homo sapiens 9 Ile ProGln Phe Val 1 5 10 18 DNA Homo sapiens 10 gaaattccac aatttgtg 18 11 6PRT Homo sapiens 11 Glu Ile Pro Gln Phe Val 1 5 12 2466 PRT Homo sapiens12 Met His Val Ser Leu Ala Glu Ala Leu Glu Val Arg Gly Gly Pro Leu 1 510 15 Gln Glu Glu Glu Ile Trp Ala Val Leu Asn Gln Ser Ala Glu Ser Leu 2025 30 Gln Glu Leu Phe Arg Lys Val Ser Leu Ala Asp Pro Ala Ala Leu Gly 3540 45 Phe Ile Ile Ser Pro Trp Ser Leu Leu Leu Leu Pro Ser Gly Ser Val 5055 60 Ser Phe Thr Asp Glu Asn Ile Ser Asn Gln Asp Leu Arg Ala Phe Thr 6570 75 80 Ala Pro Glu Val Leu Gln Asn Gln Ser Leu Thr Ser Leu Ser Asp Val85 90 95 Glu Lys Ile His Ile Tyr Ser Leu Gly Met Thr Leu Tyr Trp Gly Ala100 105 110 Asp Tyr Glu Val Pro Gln Ser Gln Pro Ile Lys Leu Gly Asp HisLeu 115 120 125 Asn Ser Ile Leu Leu Gly Met Cys Glu Asp Val Ile Tyr AlaArg Val 130 135 140 Ser Val Arg Thr Val Leu Asp Ala Cys Ser Ala His IleArg Asn Ser 145 150 155 160 Asn Cys Ala Pro Ser Phe Ser Tyr Val Lys HisLeu Val Lys Leu Val 165 170 175 Leu Gly Asn Leu Ser Gly Thr Asp Gln LeuSer Cys Asn Ser Glu Gln 180 185 190 Lys Pro Asp Arg Ser Gln Ala Ile ArgAsp Arg Leu Arg Gly Lys Gly 195 200 205 Leu Pro Thr Gly Arg Ser Ser ThrSer Asp Val Leu Asp Ile Gln Lys 210 215 220 Pro Pro Leu Ser His Gln ThrPhe Leu Asn Lys Gly Leu Ser Lys Ser 225 230 235 240 Met Gly Phe Leu SerIle Lys Asp Thr Gln Asp Glu Asn Tyr Phe Lys 245 250 255 Asp Ile Leu SerAsp Asn Ser Gly Arg Glu Asp Ser Glu Asn Thr Phe 260 265 270 Ser Pro TyrGln Phe Lys Thr Ser Gly Pro Glu Lys Lys Pro Ile Pro 275 280 285 Gly IleAsp Val Leu Ser Lys Lys Lys Ile Trp Ala Ser Ser Met Asp 290 295 300 LeuLeu Cys Thr Ala Asp Arg Asp Phe Ser Ser Gly Glu Thr Ala Thr 305 310 315320 Tyr Arg Arg Cys His Pro Glu Ala Val Thr Val Arg Thr Ser Thr Thr 325330 335 Pro Arg Lys Lys Glu Ala Arg Tyr Ser Asp Gly Ser Ile Ala Leu Asp340 345 350 Ile Phe Gly Pro Gln Lys Met Asp Pro Ile Tyr His Thr Arg GluLeu 355 360 365 Pro Thr Ser Ser Ala Ile Ser Ser Ala Leu Asp Arg Ile ArgGlu Arg 370 375 380 Gln Lys Lys Leu Gln Val Leu Arg Glu Ala Met Asn ValGlu Glu Pro 385 390 395 400 Val Arg Arg Tyr Lys Thr Tyr His Gly Asp ValPhe Ser Thr Ser Ser 405 410 415 Glu Ser Pro Ser Ile Ile Ser Ser Glu SerAsp Phe Arg Gln Val Arg 420 425 430 Arg Ser Glu Ala Ser Lys Arg Phe GluSer Ser Ser Gly Leu Pro Gly 435 440 445 Val Asp Glu Thr Leu Ser Gln GlyGln Ser Gln Arg Pro Ser Arg Gln 450 455 460 Tyr Glu Thr Pro Phe Glu GlyAsn Leu Ile Asn Gln Glu Ile Met Leu 465 470 475 480 Lys Arg Gln Glu GluGlu Leu Met Gln Leu Gln Ala Lys Met Ala Leu 485 490 495 Arg Gln Ser ArgLeu Ser Leu Tyr Pro Gly Asp Thr Ile Lys Ala Ser 500 505 510 Met Leu AspIle Thr Arg Asp Pro Leu Arg Glu Ile Ala Leu Glu Thr 515 520 525 Ala MetThr Gln Arg Lys Leu Arg Asn Phe Phe Gly Pro Glu Phe Val 530 535 540 LysMet Thr Ile Glu Pro Phe Ile Ser Leu Asp Leu Pro Arg Ser Ile 545 550 555560 Leu Thr Lys Lys Gly Lys Asn Glu Asp Asn Arg Arg Lys Val Asn Ile 565570 575 Met Leu Leu Asn Gly Gln Arg Leu Glu Leu Thr Cys Asp Thr Lys Thr580 585 590 Ile Cys Lys Asp Val Phe Asp Met Val Val Ala His Ile Gly LeuVal 595 600 605 Glu His His Leu Phe Ala Leu Ala Thr Leu Lys Asp Asn GluTyr Phe 610 615 620 Phe Val Asp Pro Asp Leu Lys Leu Thr Lys Val Ala ProGlu Gly Trp 625 630 635 640 Lys Glu Glu Pro Lys Lys Lys Thr Lys Ala ThrVal Asn Phe Thr Leu 645 650 655 Phe Phe Arg Ile Lys Phe Phe Met Asp AspVal Ser Leu Ile Gln His 660 665 670 Thr Leu Thr Cys His Gln Tyr Tyr LeuGln Leu Arg Lys Asp Ile Leu 675 680 685 Glu Glu Arg Met His Cys Asp AspGlu Thr Ser Leu Leu Leu Ala Ser 690 695 700 Leu Ala Leu Gln Ala Glu TyrGly Asp Tyr Gln Pro Glu Val His Gly 705 710 715 720 Val Ser Tyr Phe ArgMet Glu His Tyr Leu Pro Ala Arg Val Met Glu 725 730 735 Lys Leu Asp LeuSer Tyr Ile Lys Glu Glu Leu Pro Lys Leu His Asn 740 745 750 Thr Tyr ValGly Ala Ser Glu Lys Glu Thr Glu Leu Glu Phe Leu Lys 755 760 765 Val CysGln Arg Leu Thr Glu Tyr Gly Val His Phe His Arg Val His 770 775 780 ProGlu Lys Lys Ser Gln Thr Gly Ile Leu Leu Gly Val Cys Ser Lys 785 790 795800 Gly Val Leu Val Phe Glu Val His Asn Gly Val Arg Thr Leu Val Leu 805810 815 Arg Phe Pro Trp Arg Glu Thr Lys Lys Ile Ser Phe Ser Lys Lys Lys820 825 830 Ile Thr Leu Gln Asn Thr Ser Asp Gly Ile Lys His Gly Phe GlnThr 835 840 845 Asp Asn Ser Lys Ile Cys Gln Tyr Leu Leu His Leu Cys SerTyr Gln 850 855 860 His Lys Phe Gln Leu Gln Met Arg Ala Arg Gln Ser AsnGln Asp Ala 865 870 875 880 Gln Asp Ile Glu Arg Ala Ser Phe Arg Ser LeuAsn Leu Gln Ala Glu 885 890 895 Ser Val Arg Gly Phe Asn Met Gly Arg AlaIle Ser Thr Gly Ser Leu 900 905 910 Ala Ser Ser Thr Leu Asn Lys Leu AlaVal Arg Pro Leu Ser Val Gln 915 920 925 Ala Glu Ile Leu Lys Arg Leu SerCys Ser Glu Leu Ser Leu Tyr Gln 930 935 940 Pro Leu Gln Asn Ser Ser LysGlu Lys Asn Asp Lys Ala Ser Trp Glu 945 950 955 960 Glu Lys Pro Arg GluMet Ser Lys Ser Tyr His Asp Leu Ser Gln Ala 965 970 975 Ser Leu Tyr ProHis Arg Lys Asn Val Ile Val Asn Met Glu Pro Pro 980 985 990 Pro Gln ThrVal Ala Glu Leu Val Gly Lys Pro Ser His Gln Met Ser 995 1000 1005 ArgSer Asp Ala Glu Ser Leu Ala Gly Val Thr Lys Leu Asn Asn Ser 1010 10151020 Lys Ser Val Ala Ser Leu Asn Arg Ser Pro Glu Arg Arg Lys His Glu1025 1030 1035 1040 Ser Asp Ser Ser Ser Ile Glu Asp Pro Gly Gln Ala TyrVal Leu Asp 1045 1050 1055 Val Leu His Lys Arg Trp Ser Ile Val Ser SerPro Glu Arg Glu Ile 1060 1065 1070 Thr Leu Val Asn Leu Lys Lys Asp AlaLys Tyr Gly Leu Gly Phe Gln 1075 1080 1085 Ile Ile Gly Gly Glu Lys MetGly Arg Leu Asp Leu Gly Ile Phe Ile 1090 1095 1100 Ser Ser Val Ala ProGly Gly Pro Ala Asp Phe His Gly Cys Leu Lys 1105 1110 1115 1120 Pro GlyAsp Arg Leu Ile Ser Val Asn Ser Val Ser Leu Glu Gly Val 1125 1130 1135Ser His His Ala Ala Ile Glu Ile Leu Gln Asn Ala Pro Glu Asp Val 11401145 1150 Thr Leu Val Ile Ser Gln Pro Lys Glu Lys Ile Ser Lys Val ProSer 1155 1160 1165 Thr Pro Val His Leu Thr Asn Glu Met Lys Asn Tyr MetLys Lys Ser 1170 1175 1180 Ser Tyr Met Gln Asp Ser Ala Ile Asp Ser SerSer Lys Asp His His 1185 1190 1195 1200 Trp Ser Arg Gly Thr Leu Arg HisIle Ser Glu Asn Ser Phe Gly Pro 1205 1210 1215 Ser Gly Gly Leu Arg GluGly Ser Leu Ser Ser Gln Asp Ser Arg Thr 1220 1225 1230 Glu Ser Ala SerLeu Ser Gln Ser Gln Val Asn Gly Phe Phe Ala Ser 1235 1240 1245 His LeuGly Asp Gln Thr Trp Gln Glu Ser Gln His Gly Ser Pro Ser 1250 1255 1260Pro Ser Val Ile Ser Lys Ala Thr Glu Lys Glu Thr Phe Thr Asp Ser 12651270 1275 1280 Asn Gln Ser Lys Thr Lys Lys Pro Gly Ile Ser Asp Val ThrAsp Tyr 1285 1290 1295 Ser Asp Arg Gly Asp Ser Asp Met Asp Glu Ala ThrTyr Ser Ser Ser 1300 1305 1310 Gln Asp His Gln Thr Pro Lys Gln Glu SerSer Ser Ser Val Asn Thr 1315 1320 1325 Ser Asn Lys Met Asn Phe Lys ThrPhe Ser Ser Ser Pro Pro Lys Pro 1330 1335 1340 Gly Asp Ile Phe Glu ValGlu Leu Ala Lys Asn Asp Asn Ser Leu Gly 1345 1350 1355 1360 Ile Ser ValThr Gly Gly Val Asn Thr Ser Val Arg His Gly Gly Ile 1365 1370 1375 TyrVal Lys Ala Val Ile Pro Gln Gly Ala Ala Glu Ser Asp Gly Arg 1380 13851390 Ile His Lys Gly Asp Arg Val Leu Ala Val Asn Gly Val Ser Leu Glu1395 1400 1405 Gly Ala Thr His Lys Gln Ala Val Glu Thr Leu Arg Asn ThrGly Gln 1410 1415 1420 Val Val His Leu Leu Leu Glu Lys Gly Gln Ser ProThr Ser Lys Glu 1425 1430 1435 1440 His Val Pro Val Thr Pro Gln Cys ThrLeu Ser Asp Gln Asn Ala Gln 1445 1450 1455 Gly Gln Gly Pro Glu Lys ValLys Lys Thr Thr Gln Val Lys Asp Tyr 1460 1465 1470 Ser Phe Val Thr GluGlu Asn Thr Phe Glu Val Lys Leu Phe Lys Asn 1475 1480 1485 Ser Ser GlyLeu Gly Phe Ser Phe Ser Arg Glu Asp Asn Leu Ile Pro 1490 1495 1500 GluGln Ile Asn Ala Ser Ile Val Arg Val Lys Lys Leu Phe Ala Gly 1505 15101515 1520 Gln Pro Ala Ala Glu Ser Gly Lys Ile Asp Val Gly Asp Val IleLeu 1525 1530 1535 Lys Val Asn Gly Ala Ser Leu Lys Gly Leu Ser Gln GlnGlu Val Ile 1540 1545 1550 Ser Ala Leu Arg Gly Thr Ala Pro Glu Val PheLeu Leu Leu Cys Arg 1555 1560 1565 Pro Pro Pro Gly Val Leu Pro Glu IleAsp Thr Ala Leu Leu Thr Pro 1570 1575 1580 Leu Gln Ser Pro Ala Gln ValLeu Pro Asn Ser Ser Lys Asp Ser Ser 1585 1590 1595 1600 Gln Pro Ser CysVal Glu Gln Ser Thr Ser Ser Asp Glu Asn Glu Met 1605 1610 1615 Ser AspLys Ser Lys Lys Gln Cys Lys Ser Pro Ser Arg Arg Asp Ser 1620 1625 1630Tyr Ser Asp Ser Ser Gly Ser Gly Glu Asp Asp Leu Val Thr Ala Pro 16351640 1645 Ala Asn Ile Ser Asn Ser Thr Trp Ser Ser Ala Leu His Gln ThrLeu 1650 1655 1660 Ser Asn Met Val Ser Gln Ala Gln Ser His His Glu AlaPro Lys Ser 1665 1670 1675 1680 Gln Glu Asp Thr Ile Cys Thr Met Phe TyrTyr Pro Gln Lys Ile Pro 1685 1690 1695 Asn Lys Pro Glu Phe Glu Asp SerAsn Pro Ser Pro Leu Pro Pro Asp 1700 1705 1710 Met Ala Pro Gly Gln SerTyr Gln Pro Gln Ser Glu Ser Ala Ser Ser 1715 1720 1725 Ser Ser Met AspLys Tyr His Ile His His Ile Ser Glu Pro Thr Arg 1730 1735 1740 Gln GluAsn Trp Thr Pro Leu Lys Asn Asp Leu Glu Asn His Leu Glu 1745 1750 17551760 Asp Phe Glu Leu Glu Val Glu Leu Leu Ile Thr Leu Ile Lys Ser Glu1765 1770 1775 Lys Ala Ser Leu Gly Phe Thr Val Thr Lys Gly Asn Gln ArgIle Gly 1780 1785 1790 Cys Tyr Val His Asp Val Ile Gln Asp Pro Ala LysSer Asp Gly Arg 1795 1800 1805 Leu Lys Pro Gly Asp Arg Leu Ile Lys ValAsn Asp Thr Asp Val Thr 1810 1815 1820 Asn Met Thr His Thr Asp Ala ValAsn Leu Leu Arg Ala Ala Ser Lys 1825 1830 1835 1840 Thr Val Arg Leu ValIle Gly Arg Val Leu Glu Leu Pro Arg Ile Pro 1845 1850 1855 Met Leu ProHis Leu Leu Pro Asp Ile Thr Leu Thr Cys Asn Lys Glu 1860 1865 1870 GluLeu Gly Phe Ser Leu Cys Gly Gly His Asp Ser Leu Tyr Gln Val 1875 18801885 Val Tyr Ile Ser Asp Ile Asn Pro Arg Ser Val Ala Ala Ile Glu Gly1890 1895 1900 Asn Leu Gln Leu Leu Asp Val Ile His Tyr Val Asn Gly ValSer Thr 1905 1910 1915 1920 Gln Gly Met Thr Leu Glu Glu Val Asn Arg AlaLeu Asp Met Ser Leu 1925 1930 1935 Pro Ser Leu Val Leu Lys Ala Thr ArgAsn Asp Leu Pro Val Val Pro 1940 1945 1950 Ser Ser Lys Arg Ser Ala ValSer Ala Pro Lys Ser Thr Lys Gly Asn 1955 1960 1965 Gly Ser Tyr Ser ValGly Ser Cys Ser Gln Pro Ala Leu Thr Pro Asn 1970 1975 1980 Asp Ser PheSer Thr Val Ala Gly Glu Glu Ile Asn Glu Ile Ser Tyr 1985 1990 1995 2000Pro Lys Gly Lys Cys Ser Thr Tyr Gln Ile Lys Gly Ser Pro Asn Leu 20052010 2015 Thr Leu Pro Lys Glu Ser Tyr Ile Gln Glu Asp Asp Ile Tyr AspAsp 2020 2025 2030 Ser Gln Glu Ala Glu Val Ile Gln Ser Leu Leu Asp ValVal Asp Glu 2035 2040 2045 Glu Ala Gln Asn Leu Leu Asn Glu Asn Asn AlaAla Gly Tyr Ser Cys 2050 2055 2060 Gly Pro Gly Thr Leu Lys Met Asn GlyLys Leu Ser Glu Glu Arg Thr 2065 2070 2075 2080 Glu Asp Thr Asp Cys AspGly Ser Pro Leu Pro Glu Tyr Phe Thr Glu 2085 2090 2095 Ala Thr Lys MetAsn Gly Cys Glu Glu Tyr Cys Glu Glu Lys Val Lys 2100 2105 2110 Ser GluSer Leu Ile Gln Lys Pro Gln Glu Lys Lys Thr Asp Asp Asp 2115 2120 2125Glu Ile Thr Trp Gly Asn Asp Glu Leu Pro Ile Glu Arg Thr Asn His 21302135 2140 Glu Asp Ser Asp Lys Asp His Ser Phe Leu Thr Asn Asp Glu LeuAla 2145 2150 2155 2160 Val Leu Pro Val Val Lys Val Leu Pro Ser Gly LysTyr Thr Gly Ala 2165 2170 2175 Asn Leu Lys Ser Val Ile Arg Val Leu ArgGly Leu Leu Asp Gln Gly 2180 2185 2190 Ile Pro Ser Lys Glu Leu Glu AsnLeu Gln Glu Leu Lys Pro Leu Asp 2195 2200 2205 Gln Cys Leu Ile Gly GlnThr Lys Glu Asn Arg Arg Lys Asn Arg Tyr 2210 2215 2220 Lys Asn Ile LeuPro Tyr Asp Ala Thr Arg Val Pro Leu Gly Asp Glu 2225 2230 2235 2240 GlyGly Tyr Ile Asn Ala Ser Phe Ile Lys Ile Pro Val Gly Lys Glu 2245 22502255 Glu Phe Val Tyr Ile Ala Cys Gln Gly Pro Leu Pro Thr Thr Val Gly2260 2265 2270 Asp Phe Trp Gln Met Ile Trp Glu Gln Lys Ser Thr Val IleAla Met 2275 2280 2285 Met Thr Gln Glu Val Glu Gly Glu Lys Ile Lys CysGln Arg Tyr Trp 2290 2295 2300 Pro Asn Ile Leu Gly Lys Thr Thr Met ValSer Asn Arg Leu Arg Leu 2305 2310 2315 2320 Ala Leu Val Arg Met Gln GlnLeu Lys Gly Phe Val Val Arg Ala Met 2325 2330 2335 Thr Leu Glu Asp IleGln Thr Arg Glu Val Arg His Ile Ser His Leu 2340 2345 2350 Asn Phe ThrAla Trp Pro Asp His Asp Thr Pro Ser Gln Pro Asp Asp 2355 2360 2365 LeuLeu Thr Phe Ile Ser Tyr Met Arg His Ile His Arg Ser Gly Pro 2370 23752380 Ile Ile Thr His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr Leu Ile2385 2390 2395 2400 Cys Ile Asp Val Val Leu Gly Leu Ile Ser Gln Asp LeuAsp Phe Asp 2405 2410 2415 Ile Ser Asp Leu Val Arg Cys Met Arg Leu GlnArg His Gly Met Val 2420 2425 2430 Gln Thr Glu Asp Gln Tyr Ile Phe CysTyr Gln Val Ile Leu Tyr Val 2435 2440 2445 Leu Thr Arg Leu Gln Ala GluGlu Glu Gln Lys Gln Gln Pro Gln Leu 2450 2455 2460 Leu Lys 2465 13 322PRT Homo sapiens 13 Glu Ile Asp Lys Leu Leu Ile Ser Arg Thr Asp Gly ValAsp Val Ala 1 5 10 15 Phe Glu Arg Thr Lys Ala Trp Ser Thr Tyr Ser LysAsp Val Ile Ser 20 25 30 Tyr Val Arg Ala Arg Ile Gln Leu Glu Gln Asp HisAla Arg Lys Val 35 40 45 His Thr Leu Val Asp Thr Ser Arg Arg Asp Ile AsnLys Pro Phe Met 50 55 60 Pro Leu Arg Glu Ile Phe Glu Asn Ser Phe Asp ThrGlu Val Glu Met 65 70 75 80 Val Thr His Thr Lys Glu Thr Thr Glu His LeuLys Asp Arg Val Val 85 90 95 Glu Ala Leu Asp Ala Arg Arg Lys Glu His AspThr Val Arg Asn Ala 100 105 110 Leu Lys Val Glu Trp Thr Lys Ala Thr LysSer Leu His Asp Cys Glu 115 120 125 Glu Ser Tyr Glu Lys Ser Lys Ile ThrLeu Arg Met Arg Glu Glu Ala 130 135 140 Leu Lys Lys Ala Arg Glu Ser CysLeu Arg Thr Glu Ser Ser Pro Pro 145 150 155 160 Glu Arg Glu Ala Ser ArgArg Arg Arg Asp Leu Glu Lys Lys Ser Arg 165 170 175 Ala Val Glu Glu AlaMet Ile Lys Lys Glu Glu Ala Glu Arg Gln Val 180 185 190 Val Ser Ile ThrAla Glu Leu Arg Lys Lys Arg Arg Asp Ile Asp Lys 195 200 205 Thr Lys GluSer Val Val Glu Arg Leu Arg Glu Leu Ile Phe Gln Cys 210 215 220 Glu GlnThr Thr Lys Ala Cys Thr Val His Tyr Phe Thr Ser Leu Ala 225 230 235 240Ala Leu Trp Ala Arg Leu Pro Gly Ala Phe His Glu Leu Ser Asn Ala 245 250255 Thr Arg Asp Tyr Gln Pro Gly Thr Glu Tyr Met Ala Phe Leu Gln Thr 260265 270 Leu Pro Thr Arg Ala Ala Ser Ser Ser Ser Leu Val Arg Ser Asp Arg275 280 285 Ser Ile Asp Glu Gly Val Ala Ser Cys Asp Gly Ser Ser Ser LeuThr 290 295 300 Ser Leu Arg Arg Asn Ala Ile Asn Pro Asp Asp Glu Gly AlaLeu Pro 305 310 315 320 Asp Thr 14 451 DNA Homo sapiens 14 caaaaaagaatagtcttgaa gacaaaaaat ggccaaatat gtgtatagaa ataaaccgaa 60 ttcttctaaaaagcaaagac cttctaagac aattgccagc atcaaatttt aacagtcttc 120 atttccttatagtacatcta aagcgggtag tagatcatgc agaagaaaac aagatgaact 180 ccaaaaacttgggggtgata tttggaccaa gtctcattag gccaaggcca caactgctcc 240 tatcaccatctcctcccttg cagagtattc aaatcaagca cgcttggtag agtttctcat 300 tacttactcacagaagatct tcgatgggtc cctacagcca caagatgtta tgtgtagcat 360 aggtgttgttgatcaaggct gttttccaaa gcctctgtta tcaccagaag aaagagacat 420 tgaacgttccatgaagtcac tatttttttc t 451 15 543 DNA Homo sapiens 15 gtcaagatgaatatgagaaa gcaaagtctt ccatgtttcg tgcagaagag gagcatctgt 60 cttcaagtggcggattagca aaaaatctca acaagcaact agaaaaaaag cgaaggttgg 120 aagaggaggctctccaaaaa gtagaagaag caaatgaact ttacaaagtt tgtgtgacaa 180 atgttgaagaaagaagaaat gatctagaaa ataccaaaag agaaatttta gcacaactcc 240 ggacacttgttttccagtgt gatcttaccc ttaaagctgt aacagttaac ctcttccaca 300 tgcagcatctgcaggctgct tcccttgcag acagtttaca gtctctctgt gatagtgcca 360 aactcttatgacccaggcca agagtacagt ggaattttgt tcaaggccac aaatttcaac 420 tgaaggaaggaaaaagttga tgggaatgta aataaacatt ttaaatagtt cccaaccttc 480 agggtttgggcctgccaatt tttagggggt gttgtacggc ttcctgacag ttcttataaa 540 att 543 16347 DNA Homo sapiens unsure 321..321 n = a, c, g or t 16 agaccaagaggctgaatcag catcccaaaa gatagaagat ggtaaaaccc ctaagccact 60 ttctctgaaatctgataggt caacaaacaa tgtggagagg catactccaa ggaccaagat 120 tagacctgtaagtttgcctg tagatagact acttcttgca agtcctccta atgagagaaa 180 tggcagaaatatgggaaatg taaatttaga caagttttgc aagaatcctg cctttgaagg 240 agttaatagaaaagacgctg ctactactgt ttgttccaaa tttaatggct ttgaccagca 300 aactctacagaaaattcagg ncaaacagta tgaacaaaac agcttaa 347 17 458 DNA Mus musculus 17cttatgggag acgtaggcag tgactcgata ctacgtctac ctatttctcg agaaagtaag 60tcttttgaaa acatttctgt ggactcagtg gacttacccc atgaaaaagg aaatttttct 120cctatagaac tagacaactt gctgttaaag aacactgact ctatagagct ggctttgtcc 180tatgctaaaa catggtcaaa atataccaag aatatagtgt cgtgggttga aaaaaagctc 240aacttggaat tggagtccac tagaaatatt gtaaaattgg cagaggcaac tagatctagc 300attggtatac aagagtttat gccactgcag tctctattta ccaacgctct tctcagtgac 360atccacagca gccaccttct acaacagaca attgcagccc tccaagccaa taaatttgtg 420cagcctctac ttgggaggaa gaatgagatg gagaaaaa 458 18 308 DNA Homo sapiensunsure 20..20 n = a, c, g or t 18 caaaacagcc taactgccan gactacaatnntcatgccca gtgcactcca ggaaaaagga 60 gtgacaacaa gcctccagat tagtggggaccattctatca atgccactca acccagtaag 120 ccatatgcag agccagtcag gtcagtgagagaggcatctg agagacggtc ttcagattcc 180 taccctctcg ctcctgtcag agcacccagaacactgcagc ctcaacattg gacaacattt 240 tataaaccac atgctcccat catcagtatcagggggaatn aggagaagcc agtttcaccc 300 tcagcagc 308 19 443 DNA Homosapiens unsure 59..59 n = a, c, g or t 19 tgccactcaa cccagtaagccatatgcaga gccagtcagg tcagtgagag aggcatctna 60 gagacggtct tcagattcctaccctctcgc tcctgtcaga gcacccagaa cactgcagcc 120 tcaacattgg acaacattttataaaccaca tgctcccatc atcagtatca gggggaatga 180 ggagaagcca gcttcaccctcagcagcagt gcctcctggc acagatcacg atccccacgg 240 tctcgtggtg aagtcaatgccagacccaga caaagcatca gcttgtcctg gggcaagcaa 300 ctggtcaacc taaagaagacttttgaggga gcttgggttt gcctgatgtg gaatccaatg 360 tgttcagagg accaaggcttaaaacggatt gcaaacagtt ttgaaggacc tcggaggtgg 420 aatttccaca attttttttaggg 443 20 302 DNA Homo sapiens unsure 260..260 n = a, c, g or t 20ctactgtttg ttccaaattt aatggctttg accagcaaac tctacagaaa attcaggaca 60aacagtatga acaaaacagc ctaactgcca agactacaat gatcatgccc agtgcactcc 120aggaaaaagg agtgacaaca agcctccaga ttagtgggga ccattctatc aatgccactc 180aacccagtaa gccatatgca gagccagtca ggtcagtgag agaggcatct gagagacggt 240cttcagattc ctaccctctn gctcctgtca gagcacccag aacactgcag ccttcaacat 300 tg302 21 287 DNA Homo sapiens 21 aagctttgga aaatggaatg cacttggtagatatttcaga atttagttca catgatatct 60 gtgacgtctt gaaattatac cttcggcagctcccagaacc atttatttta tttcgattgt 120 acaaggaatt tatagacctt gcaaaagagatccaacatgt aaatgaagaa caagagacaa 180 aaaagaatag tcttgaagac aaaaaatggccaaatatgtg tatagaaata aaccgaattc 240 ttctaaaaag caaagacctt ctaagacaattgccagcatc aaatttt 287 22 332 DNA Homo sapiens unsure 261..261 n = a, c,g or t 22 cggaccaagt ctcattaggc caaggcccac aactgctcct atcaccatctcctcccttgc 60 agagtattca aatcaagcac gcttggtaga gtttctcatt acttactcacagaagatctt 120 cgatgggtcc ctacaaccac aagatgttat gtgtagcata ggtgttgttgatcaaggctg 180 ttttccaaag cctctgttat caccagaaga aagagacatt gaacgttccatgaagtcact 240 atttttttct tcaaaggaag ntatccatac ttcagagagt gaaagcaaaatttttgaanc 300 gggctacatc attttgaggn atcagnacgc at 332 23 545 DNA Musmusculus unsure 509..509 n = a, c, g or t 23 tgaccaagag catgagtcagcgtcccaaaa gatggaagat gtctgtaaaa gccccaagct 60 gctgctgctg aaatccaatagggcagcaaa cagtgtgcag agacatactc caaggaccaa 120 gatgagacct gtaagcttgcctgtagaccg gctgcttctt cttgccagtt ctcctactga 180 gagaagcagc agggatgtaggaaacgtaga ctcagacaag tttggcaaga accctgcctt 240 tgaaggactc catagaaaggacaactcaaa tactactcgc tccaaagtta atggctttga 300 ccagcaaaat gtacagaaatcctgggacac acaatatgta cggaacaatt ttactgccaa 360 gactacgatg attgttcccagtgcctaccc tgagaaggga ttgacagtaa acactgggaa 420 taacagggac catcccggcagtaaagcaca tgcagagcca gccagggctg caggagatgt 480 gtcagagcgc aggtcctctgactcctgcnc cgccactgct gtcagagcac ccagaacact 540 gcagc 545 24 261 DNAHomo sapiens unsure 218..218 n = a, c, g or t 24 ctactgtttg ttccaaatttaatggctttg accagcaaac tctacagaaa attcaggaca 60 aacagtatga acaaaacagcctaactgcca agactacaat gatcatgccc agtgcactcc 120 aggaaaaagg agtgacaacaagcctccaga ttagtgggga ccattctatc aatgccactc 180 aacccagtaa gccatatgcagagccagtca ggtcagtnag agaggcatct gagagacggt 240 cttcagattc ctaccctctc g261 25 321 DNA Homo sapiens unsure 11..11 n = a, c, g or t 25 ctcgtgcgccncttgcagag tattcaaatc aagcacgctt ggtagagttt ctcattactt 60 actcacagaagatcttcgat gggtccctac aaccacaaga tgttatgtgt agcataggtg 120 ttgttgatcaaggctgtttt ccaaagcctc tgttatcacc agaagaaaga gacattgaac 180 gttccatgaagtcactattt ttttcttcaa aggaagatat ccatacttca gagagtgaaa 240 gcaaaatttttgaacgagct acatcatttt gagggaatca gaancgcaag caaaatgcgt 300 tagggaaaatgtggatgcaa t 321 26 298 DNA Homo sapiens unsure 254..254 n = a, c, g ort 26 caaaacagcc taactgccaa gactacaatg atcatgccca gtgcactcca ggaaaaagga60 gtgacaacaa gcctccagat tagtggggac cattctatca atgccactca acccagtaag 120ccatatgcag agccagtcag gtcagtgaga gaggcatctg agagacggtc ttcagattcc 180taccctctcg ctcctgtcag agcacccaga acactgcagc ctcaacattg gacaacattt 240tataaaccac atgnctccca atcatcagtt atcnagggng gnaatgaagg gagnaagc 298 27429 DNA Mus musculus 27 tcctcgacaa caaagtacat ttgctttttg accaagagcatgagtcagcg tcccaaaaga 60 tggaagatgt ctgtaaaagc cccaagctgc tgctgctgaaatccaatagg gcagcaaaca 120 gtgtgcagag acatactcca aggaccaaga tgagacctgtaagcttgcct gtagaccggc 180 tgcttcttct tgccagttct cctactgaga gaagcagcagggatgtagga aacgtagact 240 cagacaagtt tggcaagaac cctgcctttg aaggactccatagaaaggac aactcaaata 300 ctactcgctc caaagttaat ggctttgacc agcaaaatgtacagaaatcc tgggacacac 360 aatatgtacg gaacaatttt actgccaaga ctacgatgattgttcccagt gcctaccctg 420 agaagggat 429 28 386 DNA Homo sapiens unsure4..4 n = a, c, g or t 28 caanngcann atcaaatntt aacagtctnc atttccttatagtacatcbn aagcnggtag 60 tagatcatgc aganganaac aagangaact ccaaaaactbgggggtnata tttggaccca 120 agtctcatta ggccaaggcc cacaactgct cctatcaccatctcctccct tgcagagtat 180 tcaaatcaag cacgcttggt agagtttctc attacttactcacagaagat cttcgatggg 240 tccctacagc cacaagatgt tatgtgtagc ataggtgttgttgntcaagg ctgttttcca 300 aagcctctgt tatcaccaga nganagngac attnacgntcatnngtcact atttttnctt 360 caaaggaaga tatccatact tcagng 386 29 365 DNAHomo sapiens unsure 230..230 n = a, c, g or t 29 aaaacagcct aactgccaagactacaatga tcatgcccag tgcactccag gaaaaaggag 60 tgacaacaag cctccagattagtggggacc attctatcaa tgccactcaa cccagtaagc 120 catatgcaga gccagtcaggtcagtgagag aggcatctga gagacggtct tcagattcct 180 accctctcgc tcctgtcagagcacccagga acactgcagc ctcaacattn ggacaacatt 240 ttattaaacc acatgcttcccattcattca gtattcaggg ggggatnagg gagaagccag 300 ctttcancct tcaggcaggcagtgccttct gggncaggnt tcacggtttc cccacggtcn 360 ttgtg 365 30 456 DNA Musmusculus 30 aattcgtcga caagcaatca ggcacgatta gtagagttcc ttattacttactcacagaag 60 atcttcgatg ggtccctcca gcctcaagct gttgttatat ctaacacaggtgctgtggca 120 ctcaggttga tcaaggctat cttccaaaac ctctgttatc accagatgagagagacacag 180 atcattctat gaaaccactc tttttttctt caaaggaaga tatccgtagttcagattgtg 240 agagcaaaag ttttgaatta actacatctt ttgaagaatc agaacgcagacaaaatgcat 300 tggggaaatg tgacgctcct ctcctcgaca acaaagtaca tttgctttttgaccaagagc 360 atgagtcagc gtcccaaaag atggaagatg tctgtaaaag ccccaagctgctgctgctga 420 aatccaatag ggcagcaaac agtgtgcaga ggacat 456 31 295 DNAMus musculus 31 aagccccaag ctgctgctgc tgaaatccaa tagggcagca aacagtgtgcagagacatac 60 tccaaggacc aagatgagac ctgtaagctt ccctgtagac cggctgcttcttcttgccag 120 ttctcctact gagagaagca gcagggatgt aggaaacgta gactcagacaagtttggcaa 180 gaaccctgcc tttgaaggac tccatagaaa ggacaactca aatactactcgctccaaagt 240 taatggcttt gaccagcaaa atgtacagaa atcctgggac acacaatatgtacgg 295 32 546 DNA Mus musculus 32 ggactgagga gaaaacagca ttaccctcaatagctgtacc tcctgtcctg gtgcatgctc 60 cccagatcca tgtgacaaaa tcagacccagactcagaggc cacattggct gtcctgtgca 120 gacaagtggt caacctaaag agagctctgaggagcctgcc ctgcctgagg ggactccaac 180 ttgccagaga ccacgactaa aacgaatgcagcaatttgaa gaccttgaag atgaaatccc 240 acagtttgtg taggattgtc aaaatttagatttttctgtt ttattttgtt ctgtggtgtc 300 attttgtgag agaatgtttg gacagggcccttttgtatag gattgccaaa gctgtttgtc 360 agtgtggtgt ttgttgctca tgtgggatgggagagtgtcc tgacaaggct ccgtttagcc 420 tcactggaat gatctttgaa gctgtaaagaaaaatgggtg tttttgtgtt ttttagagtt 480 gattttttcc tgaagaatga tccatttaaatgcatcactg atacatgata caatttttag 540 cagtag 546 33 328 DNA Homo sapiensunsure 157..157 n = a, c, g or t 33 gtagctgttc atgttgattt aaatgagtaaaaaatttgaa cttttaaatt caatatacac 60 ctttaatact gtgcaaatgt ttaactcctccacataggta actgagaata ttattttgga 120 aaaaatatgt aagactcata ttgtcttgatagagtgntca tctctaactc attcaaactc 180 ncttattaac catgtgccac aaacttaaatagatttcngg cattttcaga caaagcacag 240 ttgcttctag accaagaggc tgaatcagcatcccaaaaga tagaagatgg taaaacccct 300 aagccacttt ctctgaaatc cgataggg 32834 601 DNA Mus musculus unsure 493..493 n = a, c, g or t 34 gtaagtacattgtagctgtc ttcagacaca ccagaagagg gagtcagatc ttgttacgga 60 tggttgtgagccaccatgtg gttgctagga cttgaactct ggaccttcag aagagcagtc 120 gggtgctcttacccactaag ccatctcacc agcccgtgat atctttatat atgtgtgtgc 180 acacacatgtgcatgtgtgt tacttatata tgtatataaa ggggctctca agtactaccc 240 atgttctgcctgttgagtta tcaagcatat taaggtgtca ttgtttttct taaagtacac 300 atatgcatgtatattcgcta tgtctgagat agttcaaaca tcatttcaat ctctcactga 360 agttcagttagactaatatt tagttatgta cctggactta tagactctga atccagagat 420 ctagactcactgcttcctcc agtgctctct gagtcactaa acattccgaa cttccaggat 480 cttacgaaagaangaccttt aaaaaaagag taattaaaaa cttgcctaca ctaancccat 540 ggactaccccaacttggaga accatcccag gtgagaggag caaacctctg gaccctatta 600 a 601 35 613DNA Mus musculus 35 gtaagtacat tgtagctgtc ttcagacaca ccagaagagggagtcagatc ttgttacgga 60 tggttgtgag ccaccatgtg gttgctagga cttgaactctggaccttcag aagagcagtc 120 gggtgctctt acccactaag ccatctcacc agcccgtgatatctttatat atgtgtgtgc 180 acacacatgt gcatgtgtgt tacttatata tgtatataaaggggcttctc aagtactacc 240 catgttctgc ctgttgagtt atcaagcata ttaaggtgtcattgtttttc tttaaagtac 300 acatatgcat gtatattcgc tatgtctgag atagttcaaacatcatttca atctcctcac 360 tgaatgttca gttagactta atatttagtt attgtacctggacttataga ctctgaatcc 420 agagatctag actcactgct tcctccagtg ctctctgagtcactaaacat tccgaacttc 480 caggatctta cgaaagaagg gacctttaaa aaaagagtaattaaaaactt gcctacacta 540 acccattgga ctaccccaac tggagaacca tcccatgtgagaggagcaaa cctcggaccc 600 tattaatgga tac 613 36 536 DNA Mus musculus 36ttcgtcgaca aggacaaaat cagacccaga ctcagaggcc acattggctg tcctgtgcag 60acaagtggtc aacctaaaga gagctctgag gagcctgccc tgcctgagcg ggactccaac 120ttgccagagc accacgacta aaacgaatgc agcaatttga agaccttgaa gatgaaatcc 180cacagtttgt gtaggattgt caaaatttag atttttctgt tttattttgt tctgtggtgt 240cattttgtga gagaatgttt ggacagggcc cttttgtata ggattgccaa agctgtttgt 300cagtgtggtg tttgttgctc atgtgggacg ggagagtgtc ctgacaaggc tccgtttagc 360ctcactggaa tgatctttga agctgtaaag aaaaatgggt gtttttgtgt tttttagagt 420tgattttttc ctgaagaatg atccatttaa atgcatcact gatacatgat acaattttta 480gcagtaggtg caattgggga aaatcagctt tagtgtggag agtgagccca agtgca 536 37 198DNA Homo sapiens 37 cttgctgtat gtgaatccaa tgtgtcagag accaaggctaaaacgaatgc aacagtttga 60 agacctcgaa gatgaaattc acaatttgtg tagggatgtcaaatttcagg gtttttttgt 120 tgttgttgtg ttattttgtg gtattgtgct tgttttgtgacagaatgttt tgacagggcc 180 ccttttgtat aggactgc 198 38 614 DNA Musmusculus 38 cctaccttac tttctcgaga aataggtaga cgtatatcga gtcactgcctacgtctccca 60 taaggaagtt tgtgaggcta tccagagagg ttaaaaaaaa gcacagaaataaaaagaaat 120 tattatactt cttggtctct taccgtcaat ctatcgtcta taataaattgttttaagaaa 180 cacgtaagaa tcccattaca caaaccacag gcacagctcc taagagctctataaatactt 240 gcgatacagt caatagagca acacagaagg tagctcttgt cgagctgtgatggcatgtga 300 tactacctaa cagtttattt tccattatcc cgcgattcat gtaccgtacatcctcactaa 360 ggcatcagga gcactaactt caacgagagt cttcacttac agtttccaaaggtaaatgcc 420 aatgtttcaa tggaggaaaa gacttctcgg aatatatcgt ttttgttttcttcataactt 480 ctgtaaagtt cacagctata agcaaagatc agttgcagta agtggagggaaaacaccttt 540 taacaccaga tttataccaa gtcatttact tcttttaatc accatggcttcaaggcacca 600 aggaggtaga ggac 614 39 508 DNA Mus musculus 39 gcaaggggcgcaggcagagc gaggaccccg ctccttctct gctctggctg agtgctgtgt 60 gccctttgaacctggccagc gctaccagga gtttgttcag gaagtggaca ctgtccacag 120 ctgctcaaacccaccgactg cggcggctgc ggggcccagc caagtgcaga gaatgtgaag 180 ccttcatggtcagcgggaca gaatgtgaag agtgcttttt gacctgtcac aagcgctgcc 240 tggagaccctcctcatcctt tgtggacacc ggcggcttcc agcccggatg tccctctttg 300 gggttgacttcctacagctc cccagagatt tccctgagga ggttcccttt gtgattacca 360 gatgcacagctgagatagag caccgtgccc tgggcttgca gggtatctat cgggtcagcg 420 ggtctcgggtacgtgtggag cggctgtgca ggcctttgag aatggccgag cactggtcga 480 gctgtccgggaactctcctc acgatatc 508

What is claimed is:
 1. An isolated polypeptide encoded by an isolatednucleic acid molecule selected from the group consisting of (a) nucleicacid molecules which hybridize under stringent conditions to a moleculeconsisting of the nucleic acid sequence of SEQ ID NO:1 and which codefor a GTPase-activating polypeptide, wherein the stringent conditionsare selected from the group consisting of (1) hybridization at 65° C. inhybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone,0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA),wherein SSC is 0.15M sodium chloride/0.15M sodium citrate, pH 7, SDS issodium dodecyl sulphate, and EDTA is ethylenediaminetetracetic acid; and(2) hybridization at 42° C. in a hybridization solution containing 50%formamide, 5×SSC (1×SSC is 15 mM sodium citrate and 150 mM sodiumchloride), 2×Denhardt's solution, 0.5% SDS, 50 mM sodium phosphate, pH6.9, and 0.1 mg/ml salmon sperm DNA, (b) nucleic acid molecules thatencode the GTPase-activating polypeptide encoded by the nucleic acidmolecules of (a), and (c) full length complements of the nucleic acidmolecules of (a) or (b).
 2. The isolated polypeptide of claim 1, whereinthe isolated polypeptide comprises a polypeptide having the sequence ofamino acids 658-898 of SEQ ID NO:2.
 3. An isolated polypeptidecomprising the sequence of amino acids 613-652 of SEQ ID NO:2.
 4. Anisolated polypeptide comprising the sequence of amino acids 193-509 ofSEQ ID NO:2.
 5. The isolated polypeptide of claim 1, wherein theisolated polypeptide consists of a functional fragment or variant of aGTPase-activating polypeptide.
 6. An isolated polypeptide comprising aPDZ domain binding site which binds the fourth PDZ domain (PDZ4) ofPTPL1, comprising an amino acid sequence selected from the groupconsisting of the sequence of SEQ ID NO:7, the sequence of SEQ ID NO:9,and the sequence of SEQ ID NO:11.
 7. The isolated polypeptide of claim6, wherein the polypeptide consists of a polypeptide selected from thegroup consisting of a polypeptide having the sequence of SEQ ID NO:7, apolypeptide having the sequence of SEQ ID NO:9, and a polypeptide havingthe sequence of SEQ ID NO:11.
 8. An isolated complex of polypeptidescomprising: a polypeptide comprising the amino acid sequence of SEQ IDNO:12 bound to the isolated polypeptide of claim 1, wherein the complexhas phosphatase and GTPase activating activities.
 9. The isolatedcomplex of polypeptides of claim 8, wherein the polypeptides consist ofthe polypeptide of SEQ ID NO:12 and the polypeptide of SEQ ID NO:2. 10.The isolated polypeptide of claim 1, wherein the isolated polypeptide isencoded by nucleotides 184-3966 of SEQ ID NO:1.
 11. An isolatedpolypeptide comprising as its carboxy terminal amino acids an amino acidsequence selected from the group consisting of the sequence of SEQ IDNO:7, the sequence of SEQ ID NO:9, and the sequence of SEQ ID NO:11.